Recombinant chimeric virus and uses thereof

ABSTRACT

This invention provides a recombinant herpesvirus of turkeys comprising a herpesvirus of turkeys viral genome which contains a foreign DNA sequence inserted within the EcoR1 #9 fragment of the herpesvirus of turkeys viral genome, and the foreign DNA sequence is capable of being expressed in a host cell infected with the herpesvirus of turkeys. This invention provides a recombinant herpesvirus of turkeys-Marek&#39;s disease virus chimera comprising a herpesvirus of turkeys unique long viral genome region and a Marek&#39;s disease virus unique short region. Lastly, this invention provides homology vectors for producing a recombinant herpesvirus of turkeys, host cells, and vaccines and methods for immunization.

This application is a continuation of U.S. Ser. No. 08/288,065, filedAug. 9, 1994, which is a continuation of U.S. Ser. No. 08/023,610 filedFeb. 26, 1993; a continuation-in-part of PCT international ApplicationNo. PCT/US93/05681, filed Jun. 14, 1993; a continuation-in-part of U.S.Ser. No. 07/898,087 filed Jun. 12, 1992, now abandoned; U.S. Ser. No.07/225,032, filed Jul. 27, 1988, now U.S. Pat. No. 5,223,424, issuedJun. 29, 1993, which is a continuation-in-part of U.S. Ser. No.07/078,519, filed Jul. 27, 1987, now abandoned, U.S. Ser. No.06/933,107, filed Nov. 20, 1986, now abandoned, U.S. Ser. No.06/902,887, filed Sep. 2, 1986, now abandoned, U.S. Ser. No. 06/823,102,filed Jan. 27, 1986, now U.S. Pat. No. 5,068,192, issued Nov. 26, 1991,and U.S. Ser. No. 06/773,430, filed Sep. 6, 1985, now U.S. Pat. No.4,877,737, issued Oct. 31, 1989; U.S. Ser. No. 07/649,380, filed Jan.31, 1991, now abandoned, which is a continuation of U.S. Ser. No.07/078,519, filed Jul. 27, 1987, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 06/993,107, filed Nov. 20, 1986,now abandoned, U.S. Ser. No. 06/902,877, filed Sep. 2, 1986, nowabandoned, U.S. Ser. No. 06/887,140, filed Jul. 17, 1986, now abandoned,U.S. Ser. No. 06/823,102, filed Jan. 27, 1986, now U.S. Pat. No.5,068,192, issued Nov. 26, 1991, and U.S. Ser. No. 06/773,430, filedSep. 6, 1985, now U.S. Pat. No. 4,877,737, issued Oct. 31, 1989; andU.S. Ser. No. 07/914,057, filed Jul. 13, 1992 abandoned, which is acontinuation of U.S. Ser. No. 07/696,262, filed Apr. 30, 1991, nowabandoned, which is a continuation of U.S. Ser. No. 06/933,107, filedNov. 20, 1986, now abandoned, which is a continuation-in-part of U.S.Ser. No. 06/773,430, filed Sep. 6, 1985, now U.S. Pat. No. 4,877,737,issued Oct. 31, 1989, and U.S. Ser. No. 06/823,102 filed Jan. 27, 1986,now U.S. Pat. No. 5,068,192.

Throughout this application various publications are referenced byArabic numerals in parenthesis. Full citations for these publicationsmay be found at the end of the specification immediately preceding theclaims. The disclosures of these publications are in their entiretyhereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

The ability to isolate DNA and clone such isolated DNA into bacterialplasmids has greatly expanded the approaches available to make viralvaccines. The methods used to make the present invention involvemodifying cloned DNA sequences from various viral pathogens of animals,by insertions, deletions, single or multiple base changes, andsubsequent insertions of these modified sequences into the genome of thevirus. One utility of the addition of a foreign sequence is achievedwhen the foreign sequence encodes a foreign protein that is expressedduring viral infection of the animal. The resulting live virus may thenbe used in a vaccine to elicit an immune response in a host animal andprovide protection to the animal against disease. A virus with thesecharacteristics is referred to as a viral vector, because it becomes aliving vector that will carry and express the foreign protein in thehost animal. In effect it becomes an elaborate delivery system for theforeign protein(s).

More specifically, the present invention relates to the use ofherpesvirus of turkeys (HVT) as a viral vector for vaccination of birdsagainst disease. The group of herpesviruses comprise various pathogenicagents that infect and cause disease in a number of target species:swine, cattle, chickens, horses, dogs, cats, etc. Each herpesvirus isspecific for its host species, but they are all related in the structureof their genomes, their mode of replication, and to some extent in thepathology they cause in the host animal and in the mechanism of the hostimmune response to the virus infection.

The application of recombinant DNA techniques to animal viruses has arelatively recent history. The first viruses to be engineered have beenthose with the smallest genomes. In the case of the papovaviruses,because these viruses are so small and cannot accommodate much extraDNA, their use in genetic engineering has been as defective replicons.Foreign gene expression from these viruses requires a wild-type helpervirus and is limited to cell culture systems. For adenoviruses, there isa small amount of nonessential DNA that can be replaced by foreignsequences. The only foreign DNA that seems to have been expressed inadenoviruses are the T-antigen genes from papovaviruses (Mansour, etal., Proc. Natl. Acad. Sci. US, 1985; Thummel, et al., Cell, 1983;Scolnick, et al., Cell, 1981; Thummel, et al., Cell, 1981), and theherpes simplex virus (HSV) thymidine kinase gene (Haj-Ahmed and Graham,J. of Virology, 1986). These publications do not identify thenonessential regions in HVT wherein foreign DNA may be inserted, nor dothey teach how to achieve the expression of foreign genes in HVT, e.g.,which promoter sequence and termination sequence to use.

Another group of viruses that have been engineered are the poxviruses.One member of this group, vaccinia, has been the subject of muchresearch on foreign gene expression. Poxviruses are large DNA-containingviruses that replicate in the cytoplasm of the infected cell. They havea structure that is unique in that they do not contain any capsid thatis based upon icosahedral symmetry or helical symmetry. The poxvirusesare most likely to have evolved from bacterial-like microorganismsthrough the loss of function and degeneration. In part due to thisuniqueness, the advances made in the genetic engineering of poxvirusescannot be directly extrapolated to other viral systems, includingherpesviruses and HVT. Vaccinia recombinant virus constructs have beenmade in a number of laboratories that express the following insertedforeign genes: HSV thymidine kinase gene (Mackett, et al., Proc. Natl.Acad. Sci. USA, 1982; Panicali and Paoletti, Proc. Natl. Acad. Sci. USA,1982, hepatitis B surface antigen (Paoletti, et al., Proc. Natl. Acad.Sci. USA, 1984; Smith et al., Nature, 1983), HSV glycoprotein D gene,influenzae hemagglutinin gene (Panicali, et al., Proc. Natl. Acad. Sci.USA, 1983; Smith, et al., Proc. Natl. Acad. Sci. USA, 1983), malariaantigen gene (Smith, et al., Science, 1984, and vesicular stomatitisglycoprotein G gent (Mackett, et al., Science, 1986). The generaloverall features of vaccinia recombinant DNA work are similar to thetechniques used for all the viruses, especially as they relate to thetechniques in reference (Maniatis, et al., Molecular Cloning, 1982).However in detail, the vaccinia techniques are not applicable toherpesviruses and HVT. The utility of vaccinia as a vaccine vector is inquestion because of its close relationship to human smallpox and itsknown pathogenicity to humans. Thus, the use of the host-specificherpesvirus HVT is a better solution to vaccination of poultry.

Among the primate herpesviruses, only HSV of humans and, to a limitedextent, herpes saimiri of monkeys have been engineered to containforeign DNA sequences. The first use of recombinant DNA to manipulateHSV involved cloning a piece of DNA from the L-S junction region intothe unique long region of HSV DNA, specifically into the thymidinekinase gene (Moccarski, et al., Cell, 1980). This insert was not aforeign piece of DNA, rather it was a naturally occurring piece ofherpesvirus DNA that was duplicated at another place in the genome. Thispiece of DNA was not engineered to specifically express a protein, andthus this work does not involve expression of protein in herpesviruses.The next manipulation of HSV involved the creation of deletions in thevirus genome by a combination of recombinant DNA techniques andthymidine kinase selection. Using this approach, the HSV alpha-22 genehas been deleted (Post, et al., Cell, 1981), and a 15,000 basepairsequence of DNA has been deleted from the internal repeat of HSV(Poffenberger, et al., Proc. Natl. Acad. Sci. USA, 1981).

The following cases involve insertion of genes that encode protein intoherpesviruses: the insertion of HSV glycoprotein C into a naturallyoccurring deletion mutant of this gene in HSV (Gibson and Spear, J. ofVirology, 1983); the insertion of glycoprotein D of HSV type 2 into HSVtype 1 (Lee, et al., Proc. Natl. Acad. Sci. USA, 1982), with nomanipulation of promoter sequences since the gene is not `foreign`; theinsertion of hepatitis B surface antigen into HSV under the control ofthe HSV ICP4 promoter (Shih, et al., Proc. Natl. Acad. Sci. USA, 1984);and the insertion of bovine growth hormone into herpes saimiri viruswith an SV40 promoter (the promoter did not work in this system and anendogenous upstream promoter served to transcribe the gene) (Desrosiers,et al., 1984). Two additional foreign genes (chicken ovalbumin gene andEpstein-Barr virus nuclear antigen) have been inserted into HSV(Arsenakis and Roizman, 1984), and glycoprotein X of pseudorabies virushas been inserted into HSV (Post, et al., 1985).

These cases of deletion or insertion of genes into herpesvirusesdemonstrate that it is possible to genetically engineer herpesvirusgenomes by recombinant DNA techniques. The methods that have been usedto insert genes involve homologous recombination between the viral DNAcloned in plasmids and purified viral DNA transfected into the sameanimal cell. However, the extent to which one can generalize thelocation of the deletion and the sites for insertion of foreign genes isnot known from these previous studies.

One object of the present invention is a vaccine for Marek's disease.Marek's disease virus (MDV) is the causative agent of Marek's diseasewhich encompasses fowl paralysis, a common lymphoproliferative diseaseof chickens. The disease occurs most commonly in young chickens between2 and 5 months of age. The prominent clinical signs are progressiveparalysis of one or more of the extremities, incoordination due toparalysis of legs, drooping of the limb due to wing involvement, and alowered head position due to involvement of the neck muscles. In acutecases, severe depression may result. In the case of highly oncogenicstrains, there is characteristic bursal and thymic atrophy. In addition,there are lymphoid tumors affecting the gonads, lungs, liver, spleen,kidney and thymus (Mohanty and Dutta, 1981).

Most chickens are vaccinated against MDV at one day of age to protectthe bird against MDV for life. Prior to the present invention, theprincipal vaccination method for MDV involved using naturally occurringstrains of turkey herpesvirus (HVT). It would be advantageous toincorporate other antigens into this vaccination at one day of age, butefforts to combine conventional vaccines have not proven satisfactory todate due to competition and immunosuppression between pathogens. Themultivalent HVT-based vaccines engineered in this invention represent anovel way to simultaneously vaccinate against a number of differentpathogens. For the first time, a recombinant HVT with a foreign geneinserted into a non-essential region of the HVT genome is disclosed.

The types of genetic engineering that have been performed on theseherpesviruses consist of cloning parts of the virus DNA into plasmids inbacteria, reconstructuring the virus DNA while in the cloned state sothat the DNA contains deletions of certain sequences, and furthermoreadding foreign DNA sequences either in place of the deletions or atsites removed from the deletions.

A foreign gene of interest targeted for insertion into the genome of HVTmay be obtained from any pathogenic organism of interest. Typically, thegene of interest will be derived from pathogens that in poultry causediseases that have an economic impact on the poultry industry. The genesmay be derived from organisms for which there are existing vaccines, andbecause of the novel advantages of the vectoring technology the HVTderived vaccines will be superior. Also, the gene of interest may bederived from pathogens for which there is currently no vaccine but wherethere is a requirement for control of the disease. Typically, the geneof interest encodes immunogenic polypeptides of the pathogen, and mayrepresent surface proteins, secreted proteins and structural proteins.

A relevant avian pathogen that is a target for HVT vectoring isInfectious Laryngotracheitis virus (ILTV). ILTV is a member of theherpesviridiae family, and this pathogen causes an acute disease ofchickens which is characterized by respiratory depression, gasping andexpectoration of bloody exudate. Viral replication is limited to cellsof the respiratory tract, where in the trachea the infection gives riseto tissue erosion and hemorrhage. In chickens, no drug has beeneffective in reducing the degree of lesion formation or in decreasingclinical signs. Vaccination of birds with various modified forms of theILT virus derived by cell passage and/or tedious regimes ofadministration have conferred acceptable protection in susceptiblechickens. Because of the degree of attenuation of current ILT vaccinescare must be taken to assure that the correct level of virus ismaintained; enough to provide protection, but not enough to causedisease in the flock.

An additional target for the HVT vectoring approach is Newcastledisease, an infectious, highly contagious and debilitating disease thatis caused by the Newcastle disease virus (NDV). NDV is a single-strandedRNA virus of the paramyxovirus family. The various pathotypes of NDV(velogenic, mesogenic, lentogenic) differ with regard to the severity ofthe disease, the specificity and symptoms, but most types seem to infectthe respiratory system and the nervous system. NDV primarily infectschickens, turkeys and other avian species. Historically vaccination hasbeen used to prevent disease, but because of maternal antibodyinterferences, life-span of the bird and route of administration, theproducer needs to adapt immunization protocols to fit specific needs.

The therapeutic agent that is delivered by a viral vector of the presentinvention must be a biological molecule that is a by-product of swinepoxvirus replication. This limits the therapeutic agent in the firstanalysis to either DNA, RNA, or protein. There are examples oftherapeutic agents from each of these classes of compounds in the formof anti-sense DNA, anti-sense RNA (S. Joshi, et al., J. of Virology,1991), ribozymes (M. Wachsman, et al., J. of General Virology, 1989),suppressor tRNAs (R. A. Bhat, et al., Nucleic Acids Research, 1989),interferon-inducing double stranded RNA and numerous examples of proteintherapeutics, from hormones, e.g., insulin, to lymphokines, e.g.,interferons and interleukins, to naturals opiates. The discovery ofthese therapeutic agents and the elucidation of their structure andfunction does not make obvious the ability to use them in a viral vectordelivery system.

SUMMARY OF THE INVENTION

This invention provides a recombinant herpesvirus of turkeys comprisinga herpesvirus of turkeys viral genome which contains a foreign DNAsequence inserted within the EcoR1 #9 fragment of the herpesvirus ofturkeys viral genome, and the foreign DNA sequence is capable of beingexpressed in a host cell infected with the herpesvirus of turkeys.

Lastly, this invention provides homology vectors for producing arecombinant herpesvirus of turkeys, host cells, and vaccines and methodsfor immunization.

BRIEF DESCRIPTION OF THE FIGURES

FIGS 1A-1C

Details of HVT Construction and Map Data

FIG. 1A shows BamHI restriction fragment map of the HVT genome.Fragments are numbered in order of decreasing size; letters refer tosmall fragments whose comparative size has not been determined.

FIG. 1B shows BamHI #16 fragment of the HVT genome showing location ofβ-galactosidase gene insertion in S-HVT-001.

FIG. 1C shows BamHI #19 fragment of the HVT genome showing location ofβ-galactosidase gene insertion.

Legend: B=BamHI; X=XhoI; H=HindIII; P=PstI; S=SalI; N=NdeI; R=EcoRI.

FIGS. 2A-2D

Insertion in Plasmid 191-47

FIG. 2A contains a diagram showing the orientation of DNA fragmentsassembled in plasmid 191-47. FIGS. 2A to 2D show the sequences locatedat each of the junctions between the DNA fragments in plasmid 191-47.(SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, and 27).

FIGS. 3A-3B

Details of S-HVT-003 Construction

FIG. 3A shows restriction map of HVT DNA in the region of the BamHI #16fragment. This fragment is contained within large HindIII fragment. FIG.3A also shows the XhoI site which was first changed to an EcoRI (R) siteby use of a "linker" and standard cloning procedures. FIG. 3A also showsdetails of the construction of the beta-gal gene and IBVD gene insertedinto the BamHI #16 fragment for use in homologous recombination. Bothgenes were under the control of the PRV gX gene promoter (gX).

FIG. 3B show the S-HVT-003 genome, including the location of the twoinserted foreign genes, β-gal and IBDV.

In FIG. 3: H=HindIII; B=BamHI; X=XhoI; R=EcoRI; Xb=XbaI; Hp=HpaI;S=SmaI; UL=unique long region; US=unique short region; IR=internalrepeat region; TR=terminal repeat region.

FIG. 4

Western blot indicating the differential expression of the IBDV 32 kDantigen in cellular lysates of S-HVT-003 infected cells (32 kD present)and S-HVT-001 infected cells (32 kD negative). IBDV specificpolypeptides were identified by probing the blot with hyper-immune ratantiserum directed against denatured IBDV virions. This serum reactsprimarily with the immunodominant 32 kD antigen (IBDV VP3). The lanes onthe blot contain: 1) protein molecular weight standards, 2) uninfectedCEF cells, 3) S-HVT-001 infected CEF's, 4) 5) & 6) S-HVT-003 and 7) IBDVvirion polypeptides.

FIG. 5

Western blot indicating the differential expression of the 42 kD (VP2)antigen in cellular lysates of S-HVT-003 infected cells (42 kD present)and S-HVT-001 infected cells (42 kD negative). IBDV specificpolypeptides were identified using a VP2 specific rabit anti-peptideantiserum. The lanes contain: 1) protein molecular weight standards, 2)wild-type HVT infected CEF's, 3) S-HVT-001 infected CEF's, 4) S-HVT-003infected CEF's, 5) S-HVT-003 infected CEF's, and 6) IBDV virionpolypeptides.

FIGS. 6A-6C

Details of S-HVT-004 Construction

FIG. 6A is a restriction map of HVT DNA in the region of the BamHI #16fragment. This fragment is contained within a large HindIII fragment.Shown also is the XhoI site (X) where applicants have made theirinsertion. Before the insertion, the XhoI was first changed to EcoRI (R)site by use of a "linker" and standard cloning procedures.

FIG. 6B provides details of the construction of the β-gal gene and MDVgA gene inserted into the BamHI #16 fragment for use in homologousrecombination. Beta-gal was under the control of the PRV gX genepromoter (gX), while the MDV gA gene was under the control of its ownpromoter.

FIG. 6C is of S-HVT-004 genome showing the location of the two insertedforeign genes, β-gal and MDV gA.

In FIG. 6, H=HindIII; B=BamHI; X=XhoI; R=EcoRI; Xb=XbaI; UL=unique longregion; US=unique short region; IR=internal repeat region; TR=terminalrepeat region.

FIGS. 7A-7B

Detailed description of the β-galactosidase (lacZ) marker gene insertionin homology vector 467-22.A12. FIG. 7A shows a diagram indicating theorientation of DNA fragments assembled in the marker gene. The origin ofeach fragment is described in the Materials and Methods section. FIGS.7A and 7B show the DNA sequences located at the junctions between DNAfragments and at the ends of the marker gene (SEQ ID NOs: 28, 29, 30,31, 32, and 33). FIGS. 7A and 7B further show the restriction sites usedto generate each DNA fragment at the appropriate junction and thelocation of the lacZ gene coding region. Numbers in parenthesis ( )refer to amino acids, and restriction sites in brackets [ ] indicate theremnants of sites which were destroyed during construction. Thefollowing abbreviations are used, pseudorabies virus (PRV), lactoseoperon Z gene (lacZ), Escherichia coli (E.Coli), polyadenylation signal(pA), and glycoprotein X (gpx).

FIG. 8

BamHI, NotI restriction map of the HVT genome. The unique long (UL) andunique short (US) regions are shown. The long and short region repeatsare indicated by boxes. The BamHI fragments are numbered in decreasingorder of size. The location of probes P1-P4 are indicated. The origin ofeach probe is as follows: P1--BamHI #6, P2--BamHI #2, P3--BamHI #13, andP4--4.0 kb BgIII to StuI sub-fragment of HVT genomic XbaI fragment #5(8.0 kb).

FIG. 9

Shows the Procedure for construction of plasmid pSY229.

FIGS. 10A-10B

Detailed description of the MDV gene cassette insert in Homology Vectors456-18.18 and 456-17.22. FIG. 10A and 10B show a diagram indicating theorientation of DNA fragments assembled in the cassette and the locationof the MDV gA and gB genes. The origin of each fragment is described inthe Materials and Methods section. The sequences located at thejunctions between each fragment and at the ends of the marker gene areshown in FIGS. 10A and 10B, including junction A (SEQ ID NO: 34),junction B (SEQ ID NO: 35), and junction C (SEQ ID NO: 36). Therestriction sites used to generate each fragment are indicated at theappropriate junction. Numbers in parenthesis ( ) refer to amino acids,and restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction.

FIGS. 11A-11B

Detailed description of the HindIII fragment insert in Homology Vector556-41.5. The diagram of FIGS. 11A and 11B show the orientation of DNAfragments assembled in the cassette. The origin of each fragment isdescribed in the Materials and Methods section. FIGS. 11A and 11Bfurther show the DNA sequences located at the junctions between each DNAfragment of the plasmid and at the ends of the marker gene, includingjunction A (SEQ ID NO: 37), junction B (SEQ ID NO: 38), and junction C(SEQ ID NO: 39). The restriction sites used to generate each fragmentare indicated at the appropriate junction. The location of the MDV gDand a portion of the gI gene is also given. Numbers in parenthesis ( )refer to amino acids, and restriction sites in brackets [ ] indicate theremnants of sites which were destroyed during construction.

FIGS. 12A-12C

Detailed description of the SalI fragment insert in Homology Vector255-18.B16. FIG. 12A shows a diagram indicating the orientation of DNAfragments assembled in the cassette. The origin of each fragment isdescribed in the Materials and Methods section. FIGS. 12A to 12C furthershow the DNA sequences located at the junctions between each fragmentand at the ends of the marker gene are shown, including junction A (SEQID NO: 40), junction B (SEQ ID NO: 41), junction C (SEQ ID NO: 42),junction D (SEQ ID NO: 43), junction E (SEQ ID NO: 44), junction F(SEQID NO: 45), junction G (SEQ ID NO: 46), and junction H (SEQ ID NO: 47).The restriction sites used to generate each fragment are indicated atthe appropriate junction. The location of the NDV F and lacZ-NDV HNhybrid gene are shown. Numbers in parenthesis ( ) refer to amino acids,and restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction.

FIGS. 13A-13B

Show how the unique XhoI site of the BamHI #10 fragment of the HVTgenome was converted into a PacI site and a NotI site by insertion ofthe synthetic DNA sequence at the XhoI site (Nucleotides #1333-1338; SEQID NO. 48). FIG. 13A shows the Xho site converted into a PacI site togenerate Plasmid 654-45.1 (SEQ ID NO. 55) and FIG. 13B shows the XhoIsite converted into a NotI site to generate Plamid 686-63.A1 (SEQ ID NO.56).

FIG. 14

Restriction map and open reading frames of the sequence surrounding theinsertion site within the unique long of HVT (SEQ ID NO. 48). This mapshows the XhoI restriction site (SEQ ID NO. 48; Nucl. 1333-1338) usedfor insertion of foreign genes. Also shown are four open reading frameswithin this sequence. ORF A is interrupted by insertion of DNA into theXhoI site. The ORF A amino acid sequence (SEQ ID NO. 50; Nucl. 1402 to602; 267 amino acids) shows no significant sequence identity to anyknown amino acid sequence in the protein databases. UL 54 (SEQ ID NO.49; Nucl. 146 to 481; 112 amino acids) and UL55 (SEQ ID NO. 51; Nucl.1599 to 2135; 179 amino acids) show significant sequence identity to theherpes simplex virus type I UL54 and UL55 proteins, respectively. ORF B(SEQ ID NO. 52; Nucl. 2634 to 2308; 109 amino acids) shows nosignificant sequence identity to any known amino acid sequence in theprotein databases. Searches were performed on NCBI databases using Blastsoftware.

FIG. 15

Restriction map of cosmids 407-32.1C1, 672-01.A40, 672-07.C40, and654-45.1. The overlap of HVT genomic DNA fragments EcoRI #9 and BamHI#10 is illustrated. A unique XhoI site within the EcoRI #9 and BamHI #10fragments has been converted to a unique PacI site in Plasmid 654-45.1or a unique NotI site in Plasmid 686-63.A1.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a recombinant herpesvirus of turkeys (HVT)comprising a foreign DNA sequence inserted into a non-essential site inthe HVT genome. The foreign DNA sequence is capable of being expressedin a host cell infected with the recombinant HVT and its expression isunder the control of a promoter located upstream of the foreign DNAsequence.

As defined herein "a non-essential site in the HVT genome" means aregion in the HVT viral genome which is not necessary for the viralinfection or replication.

As defined herein, "viral genome" or "genomic DNA" means the entire DNAwhich the naturally occurring herpesvirus of turkeys contains. Asdefined herein, "foreign DNA sequence" or "gene" means any DNA or genethat is exogenous to the genomic DNA.

As defined herein, an "open reading frame" is a segment of DNA whichcontains codons that can be transcribed into RNA which can be translatedinto an amino acid sequence and which does not contain a terminationcodon.

The invention further provides several appropriate insertion sites inthe HVT genome useful for constructing the recombinant herpesvirus ofthe present invention. Insertion sites include the EcoRI #9 fragment andthe BamHI #10 fragment of the HVT genome, a preferred insertion sitewithin both of those fragments being a XhoI restriction endonuclease.

Another such site is the BamHI #16 fragment of the HVT genome. Apreferred insertion site within the BamHI #16 fragment lies within anopen reading frame encoding UL43 protein and a preferred insertion sitewithin that open reading frame in a XhoI restriction endonuclease site.

Yet another insertion site is the HVT US2 gene, with a preferredinsertion site within it being a StuI endonuclease site.

This invention provides a recombinant herpesvirus of turkeys comprisinga herpesvirus of turkeys viral genome which contains a foreign DNAsequence inserted within the EcoR1 #9 fragment of the herpesvirus ofturkeys viral genome, and the foreign DNA sequence is capable of beingexpressed in a host cell infected with the herpesvirus of turkeys.

In one embodiment, the foreign DNA sequence is inserted within an OpenReading Frame A (ORFA) of the EcoR1 #9 fragment. Insertion of foreignDNA sequences into the XhoI site of EcoR1 #9 interrupts ORFA indicatedthat the entire ORFA region is non-essential for replication of therecombinant.

For purposes of this invention, "a recombinant herpesvirus of turkeys"is a live herpesvirus of turkeys which has been generated by therecombinant methods well known to those of skill in the art, e.g., themethods set forth in DNA TRANSFECTION FOR GENERATING RECOMBINANTHERPESVIRUS in Materials and Methods, and the virus has not had geneticmaterial essential for the replication of the herpesvirus of turkeysdeleted. The purified herpesvirus of turkeys results in stable insertionof foreign DNA sequences or a gene in the EcoR1 #9 fragment or BamH1 #10fragment.

The invention further provides recombinant herpesvirus of turkeys wherethe foreign DNA sequence encodes a polypeptide which is antigenic in ananimal into which the recombinant herpesvirus is introduced.

In one embodiment the polypeptide is a detectable marker. For purposesof this invention, a "polypeptide which is a detectable marker" includesthe bimer, trimer and tetramer form of the polypeptide. E. coliβ-galactosidase is a tetramer composed of four polypeptides or monomersubunits. In one embodiment the polypeptide is E. colibeta-galactosidase. Preferably this recombinant herpesvirus of turkeysis designated S-HVT-001, S-HVT-014, or S-HVT-012.

S-HVT-012 has been deposited on Oct. 15, 1992 pursuant to the BudapestTreaty on the International Deposit of Microorganism for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR. 2382.

S-HVT-014 has been deposited on Dec. 7, 1993 pursuant to the BudapestTreaty on the International Deposit of Microorganism for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR. 2440.

In another embodiment the foreign DNA sequence encodes a cytokine. Inanother embodiment the cytokine is chicken myelomonocytic growth factor(cMGF) or chicken interferon (cIFN). In a preferred embodiment therecombinant herpesvirus of turkeys is designated S-HVT-144.

The invention further provides a recombinant herpesvirus of turkeyswhose viral genome contains foreign DNA encoding an antigenicpolypeptide which is from Marek's disease virus (MDV), Newcastle diseasevirus (NDV), infectious laryngotracheitis virus (ILTV), infectiousbronchitis virus (IBV) or infectious bursal disease virus (IBDV).

This invention provides a recombinant herpesvirus of turkeys with aforeign DNA sequence insertion in the EcoR1 #9 fragment which furthercomprises a foreign DNA sequence encoding the antigenic polypeptideselected from the group consisting of: Marek's disease virus, Newcastledisease virus, infectious laryngotracheitis virus, infectious bronchitisvirus and infectious bursal disease virus.

In one embodiment the foreign DNA sequence encoding the antigenicpolypeptide is from Marek's disease virus and encodes Marek's diseasevirus glycoprotein gA, Marek's disease virus glycoprotein gB or Marek'sdisease virus glycoprotein gD. In another embodiment the foreign DNAsequences encoding the Marek's disease virus glycoprotein gA,glycoprotein gB or glycoprotein gD are inserted into the unique StuIsite of the US2 gene coding region of the herpesvirus of turkeys.

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNA encoding antigenic polypeptide fromMarek's disease virus. Preferably, the antigenic polypeptide is Marek'sdisease virus glycoprotein gB, gA or gD.

In one embodiment a recombinant HVT containing a foreign DNA sequenceencodes IBDV VP2, MDV gA, and MDV gB. Preferably, such recombinant virusis designated S-HVT-137 and S-HVT-143.

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNA encoding Marek's disease virusglycoprotein gA and further comprising foreign DNA encoding apolypeptide which is a detectable marker. Preferably, this recombinantherpesvirus of turkeys is designated S-HVT-004.

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNA encoding Marek's disease virusglycoprotein gB. Preferably, this recombinant herpesvirus of turkeys isdesignated S-HVT-045.

An embodiment of a recombinant HVT containing a foreign DNA sequenceencoding MDV gB is also provided and this recombinant HVT is designatedS-HVT-045. S-HVT-045 has been deposited on Oct. 15, 1992 pursuant to theBudapest Treaty on the International Deposit of Microorganism for thePurposes of Patent Procedure with the Patent Culture Depository of theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 U.S.A. under ATCC Accession No. VR. 2383.

The present invention also provides recombinant HVTs engineered tocontain more than one foreign DNA sequence encoding an MDV antigen. Forexample, a foreign DNA sequence encoding MDV gA and gB can both bevectored into the HVT genome. Furthermore, a recombinant HVT can beconstructed to include a foreign DNA sequence encoding MDV gA, gB, andgD.

Recombinant HVT designated S-HVT-046 and S-HVT-047 provide embodimentsof a recombinant HVT containing foreign DNA sequence encoding MDV gA andgB; recombinant HVT designated S-HVT-048 and S-HVT-062 provideembodiments of a recombinant HVT containing foreign DNA sequenceencoding MDV gA, gB and gD.

S-HVT-062 has been deposited on Feb. 23, 1993 pursuant to the BudapestTreaty on the International Deposit of Microorganisms for the Purposesof Patent Procedure with the Patent Culture Depository of the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852U.S.A. under ATCC Accession No. VR. 2401.

The present invention provides a recombinant HVT containing a foreignDNA sequence encoding an antigenic polypeptide from Newcastle diseasevirus (NDV). In such case, it is preferred that the antigenicpolypeptide is Newcastle disease virus fusion (F) protein or Newcastledisease virus hemagglutinin-neuraminidase (HN), or a recombinant proteincomprising E. coli β-galactosidase fused to Newcastle disease virushemagglutinin-neuraminidase (HN). One example of such a virus isdesignated S-HVT-007.

The present invention also provides recombinant HVTs engineered tocontain one or more foreign DNA sequence encoding an antigenicpolypeptide form MDV as well as one or more foreign DNA sequencesencoding an antigenic polypeptide from NDV. Preferably, the MDVantigenic polypeptide is MDV gB, gD, or gA and the NDV F or HN.

In one embodiment of the invention, the recombinant HVT contains foreignDNA sequence encoding MDV gB, MDV gA and NDV F. Preferably, this HVT isdesignated S-HVT-048.

In one embodiment of the invention, the recombinant HVT contains foreignDNA sequence encoding MDV gB, MDV gA and NDV HN. Preferably, this HVT isdesignated S-HVT-049.

For example, a foreign DNA sequence encoding MDV gA and gB can both bevectored into the HVT genome. Furthermore, a recombinant HVT can beconstructed to include a foreign DNA sequence encoding MDV gA, gB, andgD.

Further, in another embodiment the foreign DNA sequence encoding theantigenic polypeptide is from Newcastle disease virus and encodesNewcastle disease virus fusion protein or Newcastle disease virushemagglutinin-neuraminidase. In another embodiment the foreign DNAsequences encoding the Newcastle disease virus fusion protein orNewcastle disease virus hemagglutinin-neuraminidase are inserted into aXhoI site in EcoR1 #9 of the unique long region of the herpesvirus ofturkeys. In a preferred embodiment the recombinant herpesvirus ofturkeys is designated S-HVT-136.

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNA encoding antigenic polypeptide fromMarek's disease virus and further comprising foreign DNA encodingantigenic polypeptide form Newcastle disease virus.

The present invention further provides a recombinant HVT which containsa foreign DNA sequence encoding an antigenic polypeptide from Marek'sdisease virus glycoprotein gB and Marek's disease virus glycoprotein gAand further comprising foreign DNA encoding Newcastle disease virusfusion (F) protein. Preferably, this recombinant herpesvirus of turkeysis designated S-HVT-048.

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNA encoding Marek's disease virusglycoprotein gB and Marek's disease virus glycoprotein gA and furthercomprising foreign DNA encoding Newcastle disease virushemagglutinin-neuraminidase (HN). Preferably, this recombinantherpesvirus of turkeys is designated S-HVT-049.

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNA encoding Marek's disease virusglycoprotein gB and Marek's disease virus glycoprotein gA and furthercomprising foreign DNa encoding Newcastle disease virus fusion (F)protein and Newcastle disease virus hemagglutinin-neuraminidase (HN).Preferably, this recombinant herpesvirus of turkeys is designatedS-HVT-050.

S-HVT-050 has been deposited on Feb. 23, 1993 pursuant to the BudapestTreaty on the International Deposit of Microorganisms for the Purpose ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR. 2400.

In yet another embodiment of the invention, the recombinant HVT containsforeign DNA sequence encoding MDV gB, MDV gA, MDV gD, NDV F and NDV HN.Preferably, such recombinant HVT is designated S-HVT-106 or S-HVT 128.

The invention further provides recombinant herpesvirus Further, in oneembodiment the foreign DNA sequence encodes the antigenic polypeptidefrom an infectious laryngotracheitis virus and encodes infectiouslaryngotracheitis virus glycoprotein gB, infectious laryngotracheitisvirus glycoprotein gI or infectious laryngotracheitis virus glycoproteingD.

In another embodiment the foreign DNA sequence encodes an antigenicpolypeptide which is derived or derivable from a group consisting of:MDV gA, MDV gB, MDV gD, NDV HN, NDV F, ILT gB, ILT gI, ILT gD, IBV, IBDVVP2, IBDV VP3, IBDV VP4, avian encephalomyelitis virus, avian reovirus,avian paramyxovirus, avian influenza virus, avian adenovirus, fowl poxvirus, avian coronavirus, avian rotavirus, chick anemia virus (agent),Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeriaspp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes,trematodes, poultry mites/lice, poultry protozoa.

The invention further provides a recombinant herpesvirus of turkeyswhich contains a foreign DNA sequence encoding an antigenic polypeptidefrom infectious laryngotracheitis virus. It is preferred that theantigenic polypeptide is ILTV glycoprotein gB, ILTV gD or ILTV gI.

Also provided are recombinant HVTs which are engineered to contain morethan one foreign DNA sequence encoding an ILTV antigen. For example,ILTV gB and gD can be vectored together into the HVT genome, so can ILTVgD and gI, and ILTV gB, gD and gI. Recombinant HVT designated S-HVT-051,S-HVT-052, and S-HVT-138 are embodiments of such recombinant virus.

The present invention also provides a recombinant HVT which containsmore than one foreign DNA sequence encoding an antigenic polypeptidefrom MDV as well as one or more foreign DNA sequences encoding anantigenic polypeptide from ILTV. Preferably, the MDV antigenicpolypeptide is MDV gB, gD or gA and the ILTV antigenic polypeptide isILTV gB, gD or gI.

In one embodiment of the invention, the recombinant HVT contains foreignDNA sequences encoding MDV gB, MDV gA, MDV gD, ILTV gD and ILTV gB.Preferably, this recombinant HVT is designated S-HVT-123.

In another embodiment of this invention, the recombinant HVT containsforeign DNA sequences encoding MDV gB, MDV gA, MDV gD, ILTV gIand ILTVgD. Preferably, this recombinant HVT is designated S-HVT-139 orS-HVT-140.

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNA encoding Marek's disease virusglycoprotein gB, Mareck's disease virus glycoprotein gA, and Marek'sdisease virus glycoprotein gD and further comprising foreign DNA whichencodes infectious laryngotracheitis virus glycoprotein gD, infectiouslaryngotracheitis virus glycoprotein gB, and E. coli β-galactosidase.Preferably, this recombinant herpesvirus of turkeys is designatedS-HVT-104.

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNa encoding infectious bronchitis virusspike protein or infectious bronchitis virus matrix protein.

The present invention further provides a recombinant HVT which containsa foreign DNA sequence encoding an antigenic polypeptide from infectiousbronchitis virus (IBV). Preferably, the antigenic polypeptide is IBVspike protein or IBV matrix protein.

The present invention also provides a recombinant HVT which contains oneor more foreign DNA sequences encoding an antigenic polypeptide from IBVas well as one or more foreign DNA sequences encoding an antigenicpolypeptide from MDV. Preferably, the IBV antigenic polypeptide is IBVspike protein or IBV matrix protein, and the MDV antigenic polypeptideis MDV gB, gD or gA. One embodiment of such recombinant virus isdesignated S-HVT-066.

The invention further provides a recombinant herpesvirus of turkeyswhose genomic DNA contains foreign DNA encoding antigenic polypeptidefrom infectious bursal disease virus and further comprising foreign DNAencoding a polypeptide which is a detectable marker.

Further, in one embodiment a foreign DNA sequence encoding the antigenicpolypeptide is from infectious bursal disease virus. In anotherembodiment the foreign DNA sequence encodes infectious bursal diseasevirus VP2 gene. In another embodiment the foreign DNA sequence encodesinfectious bursal disease virus VP3 gene. In another embodiment theforeign DNA sequence encodes infectious bursal disease virus VP4 gene.Preferably, this recombinant herpesvirus of turkeys is designatedS-HVT-003 or S-HVT-096.

Recombinant HVT designated S-HVT-003 and S-HVT-096 are each anembodiment of a recombinant HVT comprising foreign DNA sequence encodingantigenic polypeptide from IBDV and encoding a detectable marker.S-HVT-003 has been deposited on Jul. 21, 1987 pursuant to the BudapestTreaty on the International Deposit of Microorganism for the Purposes ofPatent Procedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR. 2178.

This invention provides a recombinant herpesvirus of turkeys containinga foreign DNA sequence inserted into the EcoR1 #9 fragment herpesvirusof turkeys viral genome wherein the foreign DNA sequence is from aninfectious laryngotracheitis virus and encodes infectiouslaryngotracheitis virus glycoprotein gB, or infectious laryngotracheitisvirus glycoprotein gD.

In one embodiment the foreign DNA sequence is from an infectiouslaryngotracheitis virus and encodes infectious laryngotracheitis virusglycoprotein gD, or laryngotracheitis virus glycoprotein gI.

This invention provides a recombinant herpesvirus of turkeys containinga foreign DNA sequence inserted into the EcoR1 #9 fragment herpesvirusof turkeys viral genome wherein the foreign DNA sequence is from anNewcastle disease virus and encodes a Newcastle disease vius HN orNewcastle disease virus F.

This invention provides a recombinant herpesvirus of turkeys containinga foreign DNA sequence inserted into the EcoR1 #9 fragment herpesvirusof turkeys viral genome wherein the foreign DNA sequence is from aninfectious bursal virus and encodes an infectious bursal disease virusVP2, VP3, VP4.

This invention provides a recombinant herpesvirus of turkeys containinga foreign DNA sequence inserted into the EcoR1 #9 fragment herpesvirusof turkeys viral genome wherein the foreign DNA sequence is from aninfectious bronchitis virus and encodes an infectious bronchitis virusmatrix protein.

In another embodiment a foreign DNA sequence encodes an antigenicpolypeptide which is derived or derivable from a group consisting of:MDV gA, MDV gB, MDV gD, NDV HN, NDV F, ILT gB, ILT gI, ILT gD, IBV, IBDVVP2, IBDV VPD3, IBDV VP4, avian encephalomyelitis virus, avian reovirus,avian paramyxovirus, avian influenza virus, avian adenovirus, fowl poxvirus, avian coronavirus, avian rotavirus, chick anemia virus (agent),Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeriaspp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes,trematodes, poultry mites/lice, poultry protozoa. In a preferredembodiment the recombinant herpesvirus of turkeys is designatedS-HVT-136.

Such antigenic polypeptide may be derived or derivable from thefollowing: feline pathogen, canine pathogen, equine pathogen, bovinepathogen, avian pathogen, porcine pathogen, or human pathogen.

In another embodiment, the antigenic polypeptide of a human pathogen isderived from human herpesvirus, herpes simplex virus-1, herpes simplexvirus-2, human cytomegalovirus, Epstein-Barr virus, Varicell-Zostervirus, human herpesvirus-6, human herpesvirus-7, human influenza, humanimmunodeficiency virus, rabies virus, measles virus, hepatitis B virusand hepatitis C virus. Furthermore, the antigenic polypeptide of a humanpathogen may be associated with malaria or malignant tumor from thegroup consisting of Plasmodium falciparum, Bordetella pertusis, andmalignant tumor.

The invention further provides recombinant herpes virus of turkeys whosegenomic DNA contains foreign DNA encoding Newcastle disease virus fusion(F) protein and further comprising foreign DNA encoding a recombinantprotein, wherein E. coli β-galactosidase is fused to Newcastle diseasevirus hemagglutinin-neuraminidase (HN).

The invention further provides recombinant herpesvirus of turkeys whosegenomic DNA contains foreign DNA encoding Marek's disease virusglycoprotein gB and Marek's disease virus glycoprotein gA and furthercomprising foreign DNA encoding Newcastle disease virushemagglutinin-neuraminidase (HN).

This invention provides a recombinant herpesvirus of turkeys-Marek'sdisease virus chimera comprising a herpesvirus of turkeys unique longviral genome region and a Marek's disease virus unique short region. Inone embodiment the recombinant herpesvirus of turkeys-Marek's diseasevirus chimera contains a foreign DNA sequence inserted within the EcoR1#9 fragment of the herpesvirus of turkeys viral genome, and the foreignDNA sequence capable of being expressed in a host cell infected with theherpesvirus of turkeys.

In one embodiment the recombinant herpesvirus of turkeys contains aforeign DNA sequence which encodes a polypeptide. The polypeptide may beantigenic in an animal into which the recombinant herpesvirus isintroduced.

In another embodiment the polypeptide is E. coli beta-galactosidase. Inanother embodiment the foreign DNA sequence encodes a cytokine. Inanother embodiment the cytokine is chicken mylomonocytic growth factor(cMGF) or chicken interferon (cIFN).

The invention further provides recombinant herpesvirus of turkeys wherethe foreign DNA sequence encodes a polypeptide which is antigenic in ananimal into which the recombinant herpesvirus is introduced.

Further, the recombinant herpesvirus of turkeys further comprises aforeign DNA sequence encoding the antigenic polypeptide selected fromthe group consisting of: Marek's disease virus, Newcastle disease virus,infectious laryngotracheitis virus, infectious bronchitis virus andinfectious bursal disease virus.

This invention provides a recombinant herpesvirus of turkeys wherein theforeign DNA sequence is under control of an endogenous upstreamherpesvirus promoter. In one embodiment the foreign DNA sequence isunder control of a heterologous upstream promoter. In another embodimentthe promoter is selected from PRV gX, HSV-1 alpha 4, HCMV immediateearly, MDV gA, MDV gB, MDV gD, ILT gB, BHV-1.1 VP8 and ILT gD.

This invention provides a homology vector for producing a recombinantherpesvirus of turkeys by inserting foreign DNA into the viral genome ofa herpesvirus of turkey which comprises a double-stranded DNA moleculeconsisting essentially of: a) double stranded foreign DNA not usuallypresent within the herpesvirus of turkeys viral genome; b) at one endthe foreign DNA, double-stranded herpesvirus of turkeys DNA homologousto the viral genome located at one side of the EcoR1 #9 site the codingregion of the herpesvirus of turkeys viral genome; and c) at the otherend of the foreign DNA, double-stranded herpesvirus of turkeys DNAhomologous to the viral genome located at the other side of the EcoR1 #9fragment of the coding region of the herpesvirus of turkeys viralgenome. Examples of the homology vectors are designated 751-87.A8 and761-7.A1.

In one embodiment the polypeptide is antigenic in the animal into whichthe recombinant herpesvirus of turkeys is introduced. In anotherembodiment the antigenic polypeptide is from a cytokine, Marek's diseasevirus, Newcastle disease virus, infectious laryngotracheitis virus, orinfectious bronchitis virus. In a preferred embodiment the antigenicpolypeptide is a chicken mylomonocytic growth factor (cMGF) or chickeninterferon (cIFN), infectious bursal disease virus polyprotein,infectious bursal disease virus VP2 protein, Marek's disease virusglycoprotein gB, Marek's disease virus glycoprotein gA, Marek's diseasevirus glycoprotein gD, Newcastle disease virus fusion protein, Newcastledisease virus hemagglutinin-neuraminidase, infectious laryngotracheitisvirus glycoprotein gB, infectious laryngotracheitis virus glycoproteingD, infectious bronchitis virus spike protein, or infectious bronchitisvirus matrix protein.

In another embodiment the double stranded foreign DNA sequence in thehomology vector encodes an antigenic polypeptide derived from an equinepathogen. The antigenic polypeptide of an equine pathogen can derivedfrom equine influenza virus or equine herpesvirus. Examples of suchantigenic polypeptide are equine influenza virus type A/Alaska 91neuraminidase, equine influenza virus type A/Prague 56 neuraminidase,equine influenza virus type A/Miami 63 neuraminidase, equine influenzavirus type A/Kentucky 81 neuraminidaseequine herpesvirus type 1glycoprotein B, and equine herpesvirus type 1 glycoprotein D.

In another embodiment the double stranded foreign DNA sequence of thehomology vector encodes an antigenic polypeptide derived from bovinerespiratory syncytial virus or bovine parainfluenza virus. The antigenicpolypeptide of derived from bovine respiratory syncytial virus equinepathogen can derived from equine influenza virus is bovine respiratorysyncytial virus attachment protein (BRSV G), bovine respiratorysyncytial virus fusion protein (BRSV F), bovine respiratory syncytialvirus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3fusion protein, and the bovine parainfluenza virus type 3 hemagglutininneuraminidase.

In another embodiment the double stranded foreign DNA sequence in thehomology vector encodes a cytokine capable of stimulating human immuneresponse. For example, the cytokine may be, but is not limited to,interleukin-2, interleukin-6, interleukin-12, interferons,granulocyte-macrophage colony stimulating factors, and interleukinreceptors.

In one embodiment of the invention, the double-stranded herpesvirus ofturkeys DNA is homologous to DNA sequences present within the BamHI #16fragment of the herpesvirus of turkeys genome. Preferably, thedouble-stranded herpesvirus of turkeys DNA is homologous to DNAsequences present within the open reading frame encoding UL 43 proteinof the herpesvirus of turkeys genome. Preferably, this homology vectoris designated 172-29.31.

For purposes of this invention, a "homology vector" is a plasmidconstructed to insert foreign DNA in a specific site on the genome of aherpesvirus of turkeys.

In one embodiment of the invention, the double-stranded herpesvirus ofturkeys DNA is homologous to DNA sequences present within the EcoR1 #9fragment of the herpesvirus of turkeys genome. Preferably, this homologyvector is designated 172-63.1.

In one embodiment of the invention, the double-stranded herpesvirus ofturkeys DNA is homologous to DNA sequences present within the US2 genecoding region of the herpesvirus of turkeys genome. Preferably, thishomology vector is designated 435-47.1.

In another embodiment the foreign DNA sequence encodes a screenablemarker. Examples of screenable markers, include but are not limited to:E. coli β-galactosidase or E. coli B-glucuronidase.

The invention further provides a vaccine which comprises an effectiveimmunizing amount of a recombinant herpesvirus of turkeys of the presentinvention and a suitable carrier.

This invention provides a vaccine useful for immunizing a bird againstMarek's disease virus which comprises an effective immunizing amount ofthe recombinant herpesvirus of turkeys and a suitable carrier.

This invention provides a vaccine useful for immunizing a bird againstNewcastle disease virus which comprises an effective immunizing amountof the recombinant herpesvirus of turkeys and a suitable carrier.

This invention provides a vaccine useful for immunizing a bird againstinfectious laryngotracheitis virus which comprises an effectiveimmunizing amount of the recombinant herpesvirus of turkeys and asuitable carrier.

This invention provides a vaccine useful for immunizing a bird againstinfectious bronchitis virus which comprises an effective immunizingamount of the recombinant herpesvirus of turkeys and a suitable carrier.

This invention provides a vaccine useful for immunizing a bird againstinfectious bursal disease virus which comprises an effective immunizingamount of the recombinant herpesvirus of turkeys and a suitable carrier.

This invention provides a multivalent vaccine useful for immunizing abird against Marek's disease virus and Newcastle disease virus whichcomprises an effective immunizing amount of the recombinant herpesvirusof turkeys.

This invention provides a multivalent vaccine useful for immunizing abird against Marek's disease virus and infectious laryngotracheitisvirus which comprises an effective immunizing amount of the recombinantherpesvirus of turkeys and a suitable carrier.

This invention provides a multivalent vaccine useful for immunizing abird against Marek's disease virus and infectious bronchitis virus whichcomprises an effective immunizing amount of the recombinant herpesvirusof turkeys and a suitable carrier.

This invention provides a multivalent vaccine useful for immunizing abird against Marek's disease virus and infectious bursal disease viruswhich comprises an effective immunizing amount of the recombinantherpesvirus of turkeys and a suitable carrier.

The present invention also provides a method of immunizing a fowl. Forpurposes of this invention, this includes immunizing a fowl againstinfectious bursal disease virus, Marek's disease virus, Newcastledisease virus, infectious laryngotracheitis virus, or infectiousbronchitis virus. The method comprises administering to the fowl aneffective immunizing dose of the vaccine of the present invention. Thevaccine may be administered by any of the methods well known to thoseskilled in the art, for example, by intramuscular, subcutaneous,intraperitoneal or intravenous injection. Alternatively, the vaccine maybe administered intranasally or orally.

This invention provides a host cell infected with the recombinantherpesvirus of turkey. In one embodiment the host cell is an avian cell.

For purposes of this invention, a "host cell" is a cell used topropagate a vector and its insert. Infecting the cell was accomplishedby methods well known to those skilled in the art, for example, as setforth in DNA TRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUS inMaterials and Methods. Methods for constructing, selecting and purifyingrecombinant herpesvirus of turkeys are detailed below in

This invention provides a method of distinguishing chickens or otherpoultry which are vaccinated with the above vaccine from those which areinfected with a naturally-occurring Marek's disease virus whichcomprises analyzing samples of body fluids from chickens or otherpoultry for the presence of glycoprotein gG and at least one otherantigen normally expressed in chickens or other poultry infected by anaturally-occurring Marek's disease virus, the presence of thoseantigens normally expressed in infected chickens but the absence ofglycoprotein gG being indicative of vaccination with the above vaccineand not infection with a naturally-occurring Marek's disease virus.

This invention provides a recombinant herpesvirus of turkeys whichexpresses foreign DNA sequences is useful as vaccines in avian ormammalian species including but not limited to chickens, turkeys, ducks,feline, canine, bovine, equine, and primate, including human.

This vaccine may contain either inactivated or live recombinant virus.

For purposes of this invention, an "effective immunizing amount" of therecombinant feline herpes virus of the present invention is within therange of 10³ to 10⁹ PFU/dose. In another embodiment the immunizingamount is 10⁵ to 10⁷ PFU/dose. In a preferred embodiment the immunizingamount is 10⁶ PFU/dose.

The method comprises administering to the animal an effective immunizingdose of the vaccine of the present invention. The vaccine may beadministered by any of the methods well known to those skilled in theart, for example, by intramuscular, subcutaneous, intraperitoneal orintravenous injection. Alternatively, the vaccine may be administeredintranasally or orally.

Suitable carriers for the recombinant virus are well known to thoseskilled in the art and include but are not limited to proteins, sugars,etc. One example of such a suitable carrier is a physiologicallybalanced culture medium containing one or more stabilizing agents suchas hydrolyzed proteins, lactose, etc. Preferably, the live vaccine iscreated by taking tissue culture fluids and adding stabilizing agentssuch as stabilizing, hydrolyzed proteins. Preferably, the inactivatedvaccine uses tissue culture fluids directly after inactivation of thevirus.

This invention is further illustrated in the Experimental Detailssection which follows. This section is set forth to aid in anunderstanding of the invention but is not intended to, and should not beconstrued to, limit in any way the invention as set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS

Materials and Methods

Preparation of Herpesvirus of Turkeys Stock Samples

Herpesvirus of turkeys stock samples were prepared by infecting tissueculture cells at a multiplicity of infection of 0.01 PFU/cell inDulbecco's Modified Eagle Medium (DMEM) containing 2 mM glutamine, 100units/ml penicillin, 100 units/ml streptomycin (these components areobtained from Irvine Scientific or an equivalent supplier, and hereafterare referred to as complete DME medium) plus 1% fetal bovine serum.After cytopathic effect was complete, the medium and cells wereharvested and the cells were pelleted at 3000 rpm for 5 minutes in aclinical centrifuge. Infected cells were resuspended in complete mediumcontaining 20% fetal bovine serum, 10% DMSO and stored frozen at -70° C.

Preparation of Herpesvirus of Turkey DNA

All manipulations of herpesvirus of turkey (HVT) were made using strainFC-126 (ATCC #584-C). For the preparation of HVT viral DNA from thecytoplasm of infected cells, primary chicken embryo fibroblasts wereinfected at a MOI sufficient to cause extensive cytopathic effect beforethe cells overgrew. All incubations were carried out at 39° C. in ahumidified incubator with 5% CO₂ in air. Best DNA yields were obtainedby harvesting monolayers which were maximally infected, but showingincomplete cell lysis (typically 5-7 days). Infected cells wereharvested by scraping the cells into the medium using a cell scraper(Costar brand). The cell suspension was centrifuged at 3000 rpm for 10minutes at 5° C. in a GS-3 rotor (Sorvall Instruments). The resultantpellet was resuspended in cold PBS (20 ml/Roller Bottle) and subjectedto another centrifugation for 10 minutes at 3000 rpm in the cold. Afterdecanting the PBS, the cellular pellet was resuspended in 4 ml/rollerbottle of RSB buffer (10 mM Tris pH 7.5, 1 mM EDTA, and 1.5 mM MgCl₂).NP40 (Nonidet P-40™;Sigma) was added to the sample to a finalconcentration of 0.5% minutes with occasional mixing. The sample wascentrifuged for 10 minutes at 3000 rpm in the cold to pellet the nucleiand remove cellular debris. The supernatant fluid was carefullytransferred to a 15 ml Corex centrifuge tube. Both EDTA (0.5M pH 8.0)and SDS (sodium dodecyl sulfate; stock 20%) were added to the sample tofinal concentrations of 5 mM and 1%, respectively. One hundred μl ofproteinase-K (10 mg/ml; Boehringer Mannheim) was added per 4 ml ofsample, mixed, and incubated at 45° C. for 1-2 hours. After this period,an equal volume of water-saturated phenol was added to the sample andgently mixed by hand. The sample was spun in a clinical centrifuge for 5minutes at 3000 rpm to separate the phases. NaAc was added to theaqueous phase to a final concentration of 0.3M (stock solution 3M pH5.2), and the nucleic acid precipitated at -70° C. for 30 minutes afterthe addition of 2.5 volumes of cold absolute ethanol. DNA in the samplewas pelleted by spinning for 20 minutes to 8000 rpm in an HB-4 rotor at5° C. The supernatant was carefully removed and the DNA pellet washedonce with 25 ml of 80% ethanol. The DNA pellet was dried briefly byvacuum (2-3 minutes), and resuspended in 50 μl/roller bottle of infectedcells of TE buffer (10 mM Tris pH 7.5, 1 mM EDTA). Typically, yields ofviral DNA ranged between 5-10 μg/roller bottle of infected cells. Allviral DNA was stored at approximately 10° C.

Polymerase Fill-in Reaction

DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KCl, 5mM MgCl₂, and 400 micromolar each of the four deoxynucleotides. Tenunits of Klenow DNA polymerase (BRL) were added and the reaction wasallowed to proceed for 15 minutes at room temperature. The DNA was thenphenol extracted and ethanol precipitated as above.

DNA Sequencing

Sequencing was performed using the USB Sequenase Kit and ³⁵ S-dATP(NEN). Reactions using both the dGTP mixes and the dITP mixes wereperformed to clarify areas of compression. Alternatively, compressedareas were resolved on formamide gels. Templates were double-strandedplasmid subclones or single stranded M13 subclones, and primers wereeither made to the vector just outside the insert to be sequenced, or topreviously obtained sequence. Sequence obtained was assembled andcompared using Dnastar software. Manipulation and comparison ofsequences obtained was performed with Superclone and Supersee programsfrom Coral Software.

Molecular Biological Techniques

Techniques for the manipulation of bacteria and DNA, including suchprocedures as digestion with restriction endonucleases, gelelectrophoresis, extraction of DNA from gels, ligation, phosphorylationwith kinase, treatment with phosphatase, growth of bacterial cultures,transformation of bacteria with DNA, and other molecular biologicalmethods are described by Maniatis et al (1982) and Sambrook et al(1989). The polymerase chain reaction (PCR) was used to introducerestriction sites convenient for the manipulation of various DNAs. Theprocedures used are described by Innis et al (1990). In generalamplified fragments were less than 500 base pairs in size and criticalregions of amplified fragments were confirmed by DNA sequencing. Exceptas noted, these techniques were used with minor variation.

Southern Blotting of DNA

The general procedure for Southern blotting was taken from Maniatis etal. (1982). DNA was blotted to nitrocellulose filters (S&S BA85) in20×SSC (1×ssc=0.15M NaCl, 0.015M sodium citrate, pH 7.0), andprehybridized in hybridization solution consisting of 30% formamide,1×Denhardt's solution (0.02% polyvinylpyrrolidone (PVP), 0.02% bovineserum albumin (BSA), 0.02% Ficoll), 6×SSC, 50 mM NaH₂ PO₄, pH 6.8, 200μg/ml salmon sperm DNA for 4-24 hours at 55° C. Labeled probe DNA wasadded that had been labeled by nick translation using a kit fromBethesda Research Laboratories (BRL) and one ³² P-labeled nucleotide.The probe DNA was separated from the unincorporated nucleotides by NACScolumn (BRL) or on a Sephadex G50 column (Pharmacia). After overnighthybridization at 55° C., the filter was washed once with 2×SSC at roomtemperature followed by two washes with 0.1×SSC, 0.1% sodium dodecylsulfate (SDS) for 30 minutes at 55° C. The filter was dried andautoradiographed.

cDNA Cloning Procedure

cDNA cloning refers to the methods used to convert RNA molecules intoDNA molecules following state of the art procedures. Applicants' methodsare described in (Gubler and Hoffman, 1983). Bethesda ResearchLaboratories (Gaithersburg, Md.) have designed a cDNA Cloning Kit thatis very similar to the procedures used by applicants, and contains a setof reagents and protocols that may be used to duplicate our results.

For cloning virus mRNA species, a host cell line sensitive to infectionby the virus was infected at 5-10 plaque forming units per cell. Whencytopathic effect was evident, but before total destruction, the mediumwas removed and the cells were lysed in 10 mls lysis buffer (4 Mguanidine thiocyanate, 0.1% antifoam A, 25 mM sodium citrate pH 7.0,0.5% N-lauroyl sarcosine, 0.1 M beta-metcaptoethanol). The cell lysatewas poured into a sterilized Dounce homogenizer and homogenized on ice8-10 times until the solution was homogenous. For RNA purification, 8mls of cell lysate were gently layered over 3.5 mls of CsCl solution(5.7 M CsCl, 25 mM sodium citrate pH 7.0) in Beckman SW41 centrifugetube. The samples were centrifuged for 18 hrs at 20° C. at 36000 rpm ina Beckman SW41 rotor. The tubes were put on ice and the supernatantsfrom the tubes were carefully removed by aspiration to leave the RNApellet undisturbed. The pellet was resuspended in 400 μl glass distilledwater, and 2.6 mls of guanidine solution (7.5 M guanidine-HCL, 25 mMsodium citrate pH 7.0, 5 mM dithiothreitol) were added. The 0.37 volumesof 1 M acetic acid were added, followed by 0.75 volumes of cold ethanoland the sample was put at -20° C. for 18 hrs to precipitate RNA. Theprecipitate was collected by centrifugation in a Sorvall centrifuge for10 min a 4° C. at 10000 rpm in an SS34 rotor. The pellet was dissolvedin 1.0 ml distilled water, recentrifuged at 13000 rpm, and thesupernatant saved. RNA was re-extracted from the pellet 2 more times asabove with 0.5 ml distilled water, and the supernatants were pooled. A0.1 volume of 2 M potassium acetate solution was added to the samplefollowed by 2 volumes of cold ethanol and the sample was put at -20° C.for 18 hrs. The precipitated RNA was collected by centrifugation in theSS34 rotor at 4° C. for 10 min at 10000 rpm. The pellet was dissolved in1 ml distilled water and the concentration taken by absorption atA260/280. The RNA was stored at -70° C.

mRNA containing polyadenylate tails (poly-A) was selected using oligo-dTcellulose (Pharmacia #27 5543-0). Three mg of total RNA was boiled andchilled and applied to the 100 mg oligo-dT cellulose column in bindingbuffer (0.1 M Tris pH 7.5, 0.5 M LiCl, 5 mM EDTA pH 8.0, 0.1% lithiumdodecyl sulfate). The retained poly-A RNA was eluted from the columnwith elution buffer (5 mM Tris pH 7.5, 1 nM EDTA pH 8.0, 0.1% sodiumdodecyl sulfate). This mRNA was reapplied to an oligo-dT column inbinding buffer and eluted again in elution buffer. The sample wasprecipitated with 200 mM sodium acetate and 2 volumes cold ethanol at-20° C. for 18 hrs. The RNA was resuspended in 50 μl distilled water.

Ten μg poly-A RNA was denatured in 20 mM methyl mercury hydroxide for 6min at 22° C. β-mercaptoethanol was added to 75 mM and the sample wasincubated for 5 min at 22° C. The reaction mixture for first strand cDNAsynthesis in 0.25 ml contained 1 μg oligo-dT primer (P-L Bio-chemicals)or 1 μg synthetic primer, 28 units placental ribonuclease inhibitor(Bethesda Research Labs #5518SA), 100 mM Tris pH 8.3, 140 mM KCl, 10 mMMgCl₂, 0.8 mM dATP, dCTP, dGTP, and dTTP (Pharmacia), 100 microcuries ³²p-labeled dCTP (New England Nuclear #NEG-013H), and 180 units AMVreverse transcriptase (Molecular Genetics Resources #MG 101). Thereaction was incubated at 42° C. for 90 min, and then was terminatedwith 20 mM EDTA pH 8.0. The sample was extracted with an equal volume ofphenol/chloroform (1:1) and precipitated with 2 M ammonium acetate and 2volumes of cold ethanol -20° C. for 3 hrs. After precipitation andcentrifugation, the pellet was dissolved in 100 μl distilled water. Thesample was loaded onto a 15 ml G-100 Sephadex column (Pharmacia) inbuffer (100 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 100 mM NaCl). The leadingedge of the eluted DNA fractions was pooled, and DNA was concentrated bylyophilization until the volume was about 100 μl, then the DNA wasprecipitated with ammonium acetate plus ethanol as above.

The entire first strand sample was used for second strand reaction whichfollowed the Gubler and Hoffman (1983) method except that 50 μg/mldNTP's, 5.4 units DNA polymerase I (Boerhinger Mannheim #642-711), and100 units/ml E. coli DNA ligase (New England Biolabs #205) in a totalvolume of 50 microliters were used. After second strand synthesis, thecDNA was phenol/chloroform extracted and precipitated. The DNA wasresuspended in 10 μl distilled water, treated with 1 μg RNase A for 10min at 22° C., and electrophoresed through a 1% agarose gel (Sigma TypeII agarose) in 40 mM Tris-acetate pH 6.85. The gel was stained withethidium bromide, and DNA in the expected size range was excised fromthe gel and electroeluted in 8 mM Tris-acetate pH 6.85. ElectroelutedDNA was lyophilized to about 100 microliters, and precipitated withammonium acetate and ethanol as above. The DNA was resuspended in 20 μlwater.

Oligo-dC tails were added to the DNA to facilitate cloning. The reactioncontained the DNA, 100 mM potassium cacodylate pH 7.2, 0.2 mMdithiothreitol, 2 mM CaCl₂, 80 μmoles dCTP, and 25 units terminaldeoxynucleotidyl transferase (Molecular Genetic Resources #S1001) in 50μl. After 30 min at 37° C., the reaction was terminated with 10 mM EDTA,and the sample was phenol/chloroform extracted and precipitated asabove.

The dC-tailed DNA sample was annealed to 200 ng plasmid vector pBR322that contained oligo-dG tails (Bethesda Research Labs #5355 SA/SB) in200 μl of 0.01 M Tris pH 7.5, 0.1 M NaCl, 1 mM EDTA pH 8.0 at 65° C. for2 min and then 57° C. for 2 hrs. Fresh competent E. coli DH-1 cells wereprepared and transformed as described by Hanahan (1983) using half theannealed cDNA sample in twenty 200 μl aliquots of cells. Transformedcells were plated on L-broth agar plates plus 10 μg/ml tetracycline.Colonies were screened for the presence of inserts into the ampicillingene using Ampscreen (Bethesda Research Labs #5537 UA), and the positivecolonies were picked for analysis.

DNA Transfection for Generating Recombinant Herpesvirus

The method is based upon the polybrene-DMSO procedure of Kawai andNishizawa (1984) with the following modifications. Generation ofrecombinant HVT virus is dependent upon homologous recombination betweenHVT viral DNA and the plasmid homology vector containing the desiredforeign DNA flanked by the appropriate herpesvirus cloned sequences.Transfections were carried out in 6 cm plates (Corning plastic) of 50%confluent primary chick embryo fibroblast (CEF) cells. The cells wereplated out the day before in CEF growth media (1×F10/199, 5% fetal calfserum, 2% glutamine, 1% non-essential amino acids, and 2%penicillin/streptomycin) containing 4 μg/ml polybrene (stock 4 mg/ml in1×HBSS). For cotransfections into CEF cells, 5 μg of intact HVT DNA, andsuspended in 1 ml of CEF media containing 30 μg/ml polybrene (stock 4mg/ml in 1×HBSS). The DNA-polybrene suspension (1 ml) was then added toa 6 cm plate of CEF cells from which the media had been aspirated, andincubated at 39° C. for 30 minutes. The plates were rocked periodicallyduring this time to redistribute the inoculum. After this period, 4 mlof CEF growth media was added directly to wash plate, and incubated anadditional 2.5 hours a 39° C. At this time, the media was removed fromeach plate, and the cells shocked with 2 ml of 30% DMSO (DimethylSulfoxide, J. T. Baker Chemical Co.) in 1×HBSS for 4 minutes at roomtemperature. The 30% DMSO was carefully removed and the monolayerswashed once with 1×HBSS at room temperature. The cells were thenincubated at 39° C. after the addition of 5 mls of CEF growth media. Thenext day, the media was changed to remove any last traces of DMSO and tostimulate cell growth. Cytopathic effect from the virus becomes apparentwithin 6 days. Generation of a high titer stock (80%-90% CPE) canusually be made within 1 week from this date. HVT stock samples wereprepared by resuspending the infected cells in CEF growth mediacontaining 20% fetal calf serum, 10% DMSO and stored at -70° C.

Procedure for Generating Recombinant Herpesvirus from Subgenomic DNAFragments

The ability to generate herpesviruses by cotransfection of clonedoverlapping subgenmoic fragments has been demonstrated for pseudorabiesvirus (Zijl et al., 1988). If deletions and/or insertions are engineereddirectly into the subgenomic fragments prior to the cotransfection, thisprocedure results in a high frequency of viruses containing the genomicalteration, greatly reducing the amount of screening required to purifythe recombinant virus. This procedure was used to construct recombinantHVT.

A library of subclones containing overlapping HVT subgenomic fragmentswas generated as follows. HVT DNA was obtained from the American TypeCulture Collection (FC-126("Calnek")). It was sheared and then sizeselected on a glycerol gradient as described by van Zijl et al., (1988)with 40-50 kb fragments chosen as the insert population. The pooledfractions were diluted twofold with TE, one-tenth volume of 3M NaAc and2.5 volumes of ethanol were added, and the DNA was precipitated at 30Krpm in a Beckman SW41 rotor for 1 hr. The sheared fragments were givenblunt ends by initial treatment with T4 DNA polymerase, using low DNTPconcentrations to promote 3' overhang removal, followed by treatmentwith Klenow polymerase to fill in recessed 3' ends. These insertfragments were then ligated to a pWE15 (Strategene) cosmid vector, whichhad been digested with BamHI, treated with calf intestinal phosphatase,and made blunt by treatment with Klenow polymerase. The ligated mixturewas then packaged using Gigapack XL packaging extracts (Stratagene).Ligation and packaging was as recommended by the manufacturer.

Published restriction maps for the enzymes BamHI, HindIII, and XhoIpermitted the use of subcloned fragments as specific probes to screenthe cosmid library for subclones spanning the genome. Probes weregenerated from subcloned restriction fragments. The fragments were thenlabeled using a non-radioactive system (Genius, Boehringer Mannheim).Screening was facilitated by picking colonies to media followed bygrowth overnight. Sets of five filters and a master plate were stampedfrom microtiter dish and again grown overnight. Glycerol was added tothe wells to 15% and the plates were frozen at -20° C. to provide stockcultures of each colony. Filters were BioRad Colony Lift Membranes andwere treated and hybridized per manufacturer's instructions, and washedin 0.1×SSC, 0.1% SDS, 65° C. Clones which hybridized with thenon-radioactive probe were detected according to the Genius kitdirections.

Colonies were selected for further analysis on the basis of theirhybridization to two or more of the specific probes. These were thendigested with BamHI, and compared to published maps of HVT (Buckmasteret al., 1988). The three cosmids (407-32.2C3,407-32.IG7, and 407-32.5G6)were obtained in this manner. A detailed description of each clone isgiven below. It was found that chloramphenicol amplification (Maniatiset al.,1982) was necessary to achieve reasonable yields of DNA fromthese clones. In addition, one cosmid clone (407-32.5G6) was unstableand had to be grown from the original frozen stock in order to obtainsatisfactory DNA preparations.

The pWE15 vector allows the inserts to be excised with NotI. However,four NotI sites are present in the HVT genome, so that inserts spanningthese sites cannot be excised with NotI. Two of the NotI sites arepresent in the BamHI #2 fragment of HVT, this fragment was cloneddirectly in pSP64. The other two sites are present in the unique shortregion within the BamHI #1 fragment. This fragment was cloned directlyin the pWE15 vector. The three sheared cosmids and the two BamHIfragments cover all but a small portion of the ends of the HVT genome.Because these regions are repeated in the internal portions of thegenome, all of the genetic information is available.

A StuI site within the HVT US2 gene was established as a useful site forforeign DNA insertion utilizing the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUSES (see Example 6). The HVT US2gene is located within the BamHI #1 fragment which contains five StuIsites. To facilitate the use of this site for insertion of foreign DNAby the StuI site within the US2 gene was converted to a unique HindIIIsite. This was accomplished by partially digesting the BamHI #1 subclonewith StuI, and then inserting a 10 kb fragment conferring kanomycinresistance (Neo^(R)) into the site using HindIII linkers. The kanomycinresistance gene allowed positive selection of recombinant clones. TheNeOR fragment was removed by digestion with HindIII followed byreligation generating clone 430-84.215.

DNA was prepared for reconstruction experiments by restriction digestionwith enzymes which cut the subclones outside or flanking the HVTinsertions. In some instances, one cosmid in a reconstruction was usedundigested. Digested DNAs were extracted once with phenol andprecipitated with ethanol. DNA was resuspended at a concentration of 0.5to 1 μg/ml. Viral reconstruction experiments were performed usingLipofectin (BRL) to mediate transfection. Two to three micrograms eachof subclone were added to 0.5 ml of MEM media (Earle's salts)supplemented with 1% non-essential amino acids and 2%penicillin/Streptomysin (MEM+). Controls consisted of MEM+ with no DNA,with several ug of HVT DNA, or with 4 out of 5 of the subclones.Separately, 30 μl of the Lipofectin were added to another 0.5 ml. ofMEM+. These two mixtures were then combined and incubated at RT for 15minutes.

Chick embryo fibroblast (CEF) cells were prepared for transfection inthe following manner. CEFs (Spafas) were grown in 6 well dishes at 39°C. in F10/M199 (1:1) media containing 1% non-essential amino acids, 2%penicillin/streptomycin, and 5% fetal calf serum (CEF+). Cells weretransfected at a confluence of 90-95%. For transfection, wells wereaspirated and rinsed 3 times with MEM+, and then incubated 4 hours at39° C. with the 1 ml lipofectin/DNA mixture above. One ml more of CEF+was then added to the wells, and cells were incubated overnight and fedwith CEF+. Plates were then examined daily for the appearance ofplaques.

Lipofectin with control HVT DNA resulted in the appearance of plaqueswithin 5 days. When only four of the five subclones were used, noplaques were obtained. When the five overlapping genomic fragments ofHVT were used to reconstruct the virus, plaques appeared anywhere from 5to 19 days after the initial lipofection. In the case of plaquesappearing late, plaques were not initially seen on the infectedmonolayer, and it was only after passaging the monolayer and replatingon a larger surface that plaques appeared. After passaging, plaquesgenerally appeared within 3 days. Recombinant viruses were plaquepurified approximately three and then analyzed for insertion of foreignDNAs.

Bluogal Screen for Recombinant Herpesvirus

When the foreign gene encoded the enzyme β-galactosidase, the plaquesthat contained the gene were visualized more easily. The chemicalBluogal (Bethesda Research Labs) was incorporated at the level of200-300 μg/ml into the agarose overlay during the plaque assay, and theplaques that expressed active β-galactosidase turned blue. The blueplaques were then picked and purified by further blue plaque isolations.Other foreign genes were inserted by homologous recombination such thatthey replaced the β-galactosidase gene; in this instance non-blueplaques were picked for purification of the recombinant virus.

Screen for Foreign Gene Expression in Recombinant HVT Using Black PlaqueAssays

To analyze expression of foreign antigens expressed by recombinant HVTviruses, monolayers of CEF cells are infected with recombinant HVT,overlaid with nutrient agarose media and incubated for 4-5 days at 39°C. Once plaques have developed, the agarose overlay is removed from thedish, the monolayer rinsed 1× with PBS, fixed with 100% methanol for 10minutes at room temperature and the cells air dried. After re-hydratingthe plate with PBS, the primary antibody is diluted to the appropriatedilution with PBS and incubated with the cell monolayer for 2 hours toovernight at room temperature. Unbound antibody is then removed from thecells by washing three times with PBS at room temperature. An alkalinephosphatase conjugated secondary antibody is diluted with PBS andincubated with the cells for 2 hours at room temperature. Unboundsecondary antibody is then removed by washing the cells three times withPBS at room temperature. Next, the monolayer is rinsed in colordevelopment buffer (100 mM Tris pH 9.5/100 mM NaCl/5 mM MgCl2), and thenincubated 10 minutes to overnight at room temperature with freshlyprepared substrate solution (0.3 mg/ml Nitro Blue tetrazolium+0.15 mg/ml5-Bromo-4-Chloro-3-Indolyl Phosphatase in color development buffer.)Finally, the reaction is stopped by replacing the substrate solutionwith TE (10 mM Tris, pH7.5/1 mM EDTA). Plaques expressing the correctantigen will stain black.

Plaque Hybridization Procedure for Assessing the Purity of RecombinantHVT Stocks

When no suitable immunological reagent exists to detect the presence ofa particular antigen in a recombinant HVT virus, plaque hybridizationcan be used to assess the purity of a stock. Initially, CEF cellmonolayers are infected with various dilutions of the viral stocks togive ˜50-100 plaques/10 cm.dish, overlaid with nutrient agarose mediaand incubated for 4-5 days at 39° C. Once plaque development occurs, theposition of each plaque is marked on bottom of the dish. The agaroseoverlay is then removed, the plate washed with PBS, and the remainingCEF monolayer is transferred to a NC membrane or BioRad nylon membranepre-wetted with PBS (making note of the membrane position relative tothe dish). Cells contained on the NC membranes are then lysed by placingthe membranes in 1.5 mls of 1.5M NaCl and 0.5M NaOH for five minutes.The membranes are neutralized by placing them in 1.5 mls of 3M Sodiumacetate (pH 5.2) for five minutes. DNA from the lysed cells is thenbound to the NC membranes by baking at 80° C. for one hour. After thisperiod the membranes are prehybridized in a solution containing 6×SSC,3% skim milk, 0.5% SDS, (±) salmon sperm DNA (50 μg/ml) for one hour at65° C. Radio-labeled probe DNA (alpha 32P-dCTP) is then added and themembranes incubated at 65° C. overnight (˜12 hours). After hybridizationthe NC membranes are washed two times (30 minutes each) with 2×SSC at65° C., followed by two additional washes at 65° C. with 0.5×SSC. The NCmembranes are then dried and exposed to X-ray film (Kodak X-OMAT,AR) at-70° C. for 12 hours. Positive signals are then aligned with theposition of the plaques on the dish and purity of the stock is recordedas the percentage of positive plaques over the total.

Construction of Homology Vector for Insertion of the Beta-galactosidaseGene Into HVT US2 Gene

The beta-galactosidase (lacZ) gene was inserted into the HVT EcoRI # 7fragment at the unique StuI site. The marker gene is oriented in thesame direction as the US2 gene. A detailed description of the markergene is given in FIGS. 7A and 7B. It is constructed utilizing standardrecombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al,1989), by joining restriction fragments from the following sources withthe synthetic DNA sequences indicated in FIGS. 7A and 7B. Fragment 1 isan approximately 413 base pair SalI to BamHI restriction sub-fragment ofthe PRV BamHI restriction fragment 10 (Lomniczi et al., 1984). Fragment2 is an approximately 3010 base pair BamHI to PvuII restriction fragmentof plasmid pJF751 (Ferrari et al., 1985). Fragment 3 is an approximately754 base pair NdeI to SalI restriction sub-fragment of the PRV BamHIrestriction fragment #7 (Lomniczi et al., 1984).

RNA Isolated from Concanavalin a Stimulated Chicken Spleen Cells

Chicken spleens were dissected from 3 week old chicks from SPAFAS, Inc.,washed, and disrupted through a syringe/needle to release cells Afterallowing stroma and debri to settle out, the cells were pelleted andwashed twice with PBS. The cell pellet was treated with a hypotoniclysis buffer to lyse red blood cells, and splenocytes were recovered andwashed twice with PBS. Splenocytes were resuspended at 5×10⁶ cells/ml inRPMI containing 5% FBS and 5 μg/ml Concanavalin A and incubated at 39°for 48 hours. Total RNA was isolated from the cells using guanidineisothionate lysis reagents and protocols from the Promega RNA isolationkit (Promega Corporation, Madison Wis.). 4 μg of total RNA was used ineach 1st strand reaction containing the appropriate antisense primersand AMV reverse transcriptase (Promega Corporation, Madison Wis.). cDNAsynthesis was performed in the same tube following the reversetranscriptase reaction, using the appropriate sense primers and Vent®DNA polymerase (Life Technologies, Inc. Bethesda, Md.).

Subgenomic Clone 172-07.BA2

Plasmid 172-07.BA2 was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 25,000 base pair region ofgenomic HVT DNA. It may be used in conjunction with other subgenomicclones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSFROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction ofrecombinant HVT. This plasmid may be constructed utilizing standardrecombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al,1989), by joining two restriction fragments from the following sources.The first fragment is an approximately 2999 base pair BamHI to BamHIrestriction fragment of pSP64 (Promega). The second fragment is theapproximately 25,000 base pair BamHI #2 fragment of HVT (Buckmaster etal., 1988).

Homology Vector 172-29.31

The plasmid 172-29.31 was constructed for the purpose of insertingforeign DNA into HVT. It contains a unique XhoI restriction enzyme siteinto which foreign DNA may be inserted. When a plasmid containing aforeign DNA insert at the XhoI site is used according to the DNACOTRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUSES or the PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMICFRAGMENTS a virus containing the foreign DNA will result. This plasmidmay be constructed utilizing standard recombinant DNA techniques(Maniatis et al, 1982 and Sambrook et al, 1989), by joining tworestriction fragments from the following sources. The first fragment isan approximately 2999 base pair BamHI to BamHI restriction fragment ofpSP64 (Promega). The second fragment is the approximately 3300 base pairBamHI #16 fragment of HVT (Buckmaster et al., 1988). The completesequence of the BamHI #16 fragment is given in SEQ ID NO:3. Note thatthe fragment was cloned such that the UL43 ORF is in the oppositetranscriptional orientation to the pSP64 β-lacatamase gene.

Homology Vector 172-63.1

The plasmid 172-63.1 was constructed for the purpose of insertingforeign DNA into HVT. It contains a unique XhoI restriction enzyme siteinto which foreign DNA may be inserted. When a plasmid containing aforeign DNA insert at the XhoI site is used according to the DNACOTRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUSES or the PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMICFRAGMENTS a virus containing the foreign DNA will result. This plasmidmay be constructed utilizing standard recombinant DNA techniques(Maniatis et al, 1982 and Sambrook et al, 1989), by joining tworestriction fragments from the following sources. The first fragment isan approximately 2999 base pair EcoRI to EcoRI restriction fragment ofpSP64 (Promega). The second fragment is the approximately 5500 base pairEcoRI #9 fragment of HVT. Note that the EcoRI fragment was cloned suchthat the unique XhoI site is closest to the unique HindIII site in thepSP64 vector.

Homology Vectors 255-18.B16

The plasmid 255-18.B16 was constructed for the purpose of inserting theNDV HN and F genes into HVT. The NDV HN and F genes were inserted as aSalI fragment into the homology vector 172-29.31 at the XhoI site. TheNDV HN and F genes were inserted in the same transcriptional orientationthe UL43 ORF in the parental homology vector. A detailed description ofthe SalI fragment is shown in FIGS. 12A-12C. The inserted SalI fragmentmay be constructed utilizing standard recombinant DNA techniques(Maniatis et al, 1982 and Sambrook et al, 1989), by joining restrictionfragments from the following sources with the synthetic DNA sequencesindicated in FIGS. 12A, 12B and 12C. Fragment 1 is an approximately 416base pair SalI to BamHI restriction sub-fragment of the PRV BamHIrestriction fragment 10 (Lomniczi et al., 1984). Fragment 2 is anapproximately 3009 base pair BamHI to PvuII fragment of the plasmidpJF751 (Ferrari et al., 1985). Fragment 3 is an approximately 1200 basepair AvaII to EcoRI restriction fragment of full length NDV HN cDNA.Fragment 4 is an approximately 179 base pair EcoRI to PvuII restrictionfragment of the plasmid pSP64 (Promega). Fragment 5 is an approximately357 base pair SmaI to BamHI restriction sub-fragment of the HSV-1 BamHIrestriction fragment N. Fragment 6 is an approximately 1812 base pairBamHI to PstI restriction fragment of the full length NDV F cDNA.Fragment 7 is an approximately 235 base pair PstI to ScaI restrictionfragment of the plasmid pBR322.

Subgemomic Clone 378-50.BA1

Cosmid 378-50.BA1 was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 29,500 base pair region ofgenomic HVT DNA. It may be used in conjunction with other subgenomicclones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSFROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction ofrecombinant HVT. This cosmid may be constructed by joining tworestriction fragments from the following sources. The first fragment isan approximately 8164 base pair BamHI to BamHI restriction fragment ofpWE15 (Stratagene). The second fragment is the approximately 29,500 basepair BamHI #1 fragment of HVT (Buckmaster et al., 1988).

Subgemomic Clone 407-32.1C1

Cosmid 407-32.1C1 was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 38,850 base pair region ofgenomic HVT DNA (see FIG. 8). This region includes BamHI fragments 11,7, 8, 21, 6, 18, approximately 1250 base pairs of fragment 13, andapproximately 6,700 base pairs of fragment 1. It may be used inconjunction with other subgenomic clones according to the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTSfor the construction of recombinant HVT. This cosmid maybe constructedas described above in the PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. It was isolated fromthe sheared DNA library by screening with the probes P1 and P4(described in FIG. 8). A bacterial strain containing this cosmid hasbeen deposited on Mar. 3, 1993 pursuant to the Budapest Treaty on theInternational Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. 75428.

Subgemomic Clone 407-32.2C3

Cosmid 407-32.2C3 was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 40,170 base pair region ofgenomic HVT DNA (see FIG. 8). This region includes BamHI fragments 10,14, 19, 17, 5, and approximately 2,100 base pairs of fragment 2. It maybe used in conjunction with other subgenomic clones according to thePROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPINGSUBGENOMIC FRAGMENTS for the construction of recombinant HVT. Thiscosmid may be constructed as described above in the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMICFRAGMENTS. It was isolated from the sheared DNA library by screeningwith the probes P1 and P2 (described in FIG. 8). A bacterial straincontaining this cosmid has been deposited pursuant to the BudapestTreaty on the International Deposit of Microorganisms for the Purposesof Patent Procedure with the Patent Culture Depository of the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852U.S.A. under ATCC Accession No. 75430.

Subgemomic Clone 407-32.5G6

Cosmid 407-32.5G6 was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 40,000 base pair region ofgenomic HVT DNA (see FIG. 8). This region includes BamHI fragments 9, 3,20, 12, 16, 13, approximately 1,650 base pairs of fragment 2, andapproximately 4,000 base pairs of fragment 11. It may be used inconjunction with other subgenomic clones according to the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTSfor the construction of recombinant HVT. This cosmid may be constructedas described above in the PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. It was isolated fromthe sheared DNA library by screening with the probes P2 and P3(described in FIG. 8). A bacterial strain containing this cosmid hasbeen deposited on Mar. 3, 1993 pursuant to the Budapest Treaty on theInternational Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. 75427.

Homology Vector 435-47.1

The plasmid 435-47.1 was constructed for the purpose of insertingforeign DNA into HVT. It contains a unique HindIII restriction enzymesite into which foreign DNA may be inserted. When a plasmid containing aforeign DNA insert at the HindIII site is used according to the DNACOTRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUSES or the PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMICFRAGMENTS a virus containing the foreign DNA will result. This plasmidmay be constructed utilizing standard recombinant DNA techniques(Maniatis et al, 1982 and Sambrook et al, 1989), by joining tworestriction fragments from the following sources. The first fragment isan approximately 2999 base pair EcoRI to EcoRI restriction fragment ofpSP64 (Promega). The second fragment is the approximately 7300 base pairEcoRI #7 fragment of HVT. Note that the HindIII site of the pSP64 vectorwas removed by digesting the subclone with HindIII followed by a Klenowfill in reaction and religation. A synthetic HindIII linker (CAAGCTTG)was then inserted into the unique StuI site of the EcoRI #7 fragment.

Subgemomic Clone 437-26.24

Plasmid 437-26.24 was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 13,600 base pair region ofgenomic HVT DNA. It may be used in conjunction with other subgenomicclones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSFROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction ofrecombinant HVT. This plasmid may be constructed utilizing standardrecombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al,1989), by joining two restriction fragments from the following sources.The first fragment is an approximately 2970 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). The second fragment is theapproximately 13,600 base pair BamHI to StuI sub-fragment of the BamHI#2 fragment of HVT (Buckmaster et al., 1988). Note that the BamHI #2fragment contains five StuI sites, the site utilized in this subcloningwas converted to a HindIII site as described in the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMICFRAGMENTS.

Subgemomic Clone 437-26.26

Plasmid 437-26.26 was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 15,300 base pair region ofgenomic HVT DNA. It may be used in conjunction with other subgenomicclones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSFROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction ofrecombinant HVT. This plasmid may be constructed utilizing standardrecombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al,1989), by joining two restriction fragments from the following sources.The first fragment is an approximately 2970 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). The second fragment is theapproximately 15,300 base pair BamHI to StuI sub-fragment of the BamHI#2 fragment of HVT (Buckmaster et al., 1988). Note that the BamHI #2fragment contains five StuI sites, the site utilized in this subcloningwas converted to a HindIII site as described in the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMICFRAGMENTS.

Homology Vectors 456-18.18 and 456-17.22

The plasmids 456-18.18 and 456-17.22 were constructed for the purpose ofinserting the MDV gA and gB genes into HVT. The MDV genes were insertedas a cassette into the homology vector 435-47.1 at the unique HindIIIsite. The MDV genes were inserted at the blunt ended HindIII site as ablunt ended PstI to EcoRI fragment (see FIGS. 10A and 10B). The HindIIIand EcoRI sites were blunted by the Klenow fill in reaction. The PstIsite was blunted by the T4 DNA polymerase reaction. Note that the MDVcassette was inserted in both orientations. Plasmid 456-18.18 containsthe MDV genes inserted in the opposite transcriptional orientation tothe US2 gene in the parental homology vector. Plasmid 456-17.22 containsthe MDV genes inserted in the same transcriptional orientation as theUS2 gene in the parental homology vector. A detailed description of theMDV cassette is given in FIGS. 10A and 10B. It may be constructedutilizing standard recombinant DNA techniques (Maniatis et al, 1982 andSambrook et al, 1989), by joining restriction fragments from thefollowing sources with the synthetic DNA sequences indicated in FIGS.10A and 10B. Fragment 1 is an approximately 2178 base pair PvuII toEcoRV restriction sub-fragment of the MDV EcoRI 6.9 KB genomicrestriction fragment (Ihara et al., 1989). Fragment 2 is anapproximately 3898 base pair SalI to EcoRI genomic MDV fragment (Ross,et al., 1989).

Homology Vector 528-03.37

The plasmid 528-03.37 was constructed for the purpose of inserting theinfectious laryngotracheitis (ILT) virus gD gene into HVT. The gD genefollowed by the PRV gX poly adenylation signal was inserted as acassette into the homology vector 435-47.1 at the unique HindIII site.The cassette may be constructed utilizing standard recombinant DNAtechniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joiningrestriction fragments from the following sources. The first fragment isan approximately 2060 base pair EcoRI to BclI restriction sub-fragmentof the ILT KpnI genomic restriction fragment #8 (10.6 KB). The secondfragment is an approximately 754 base pair NdeI to SalI restrictionsub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi et al.,1984). Note that the fragments are oriented such that BclI and NdeIsites are contiguous.

Homology Vector 528-11.43

The plasmid 528-11.43 was constructed for the purpose of inserting theinfectious laryngotracheitis (ILT) virus gB gene (A. M. Grifin, 1991)into HVT. The gB gene was inserted as an EcoRI fragment into thehomology vector 435-47.1 at the unique HindIII site. The gB gene wasinserted at the blunt ended HindIII site as a blunt ended EcoRIfragment. The HindIII and EcoRI sites were blunted by the Klenow fill inreaction. The gB gene was inserted in the same transcriptionalorientation as the US2 gene in the parental homology vector. The EcoRIfragment may be obtained as a 3.0 KB ILT virus genomic fragment.

Homology Vector 518-46.B3

The plasmid 518-46.B3 was constructed for the purpose of insertingforeign DNA into HVT. It contains a unique HindIII restriction enzymesite into which foreign DNA may be inserted. When a plasmid containing aforeign DNA insert at the HindIII site is used according to the DNACOTRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUSES or the PROCEDUREFOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMICFRAGMENTS a virus containing the foreign DNA will result. This plasmidmay be constructed utilizing standard recombinant DNA techniques(Maniatis et al, 1982 and Sambrook et al, 1989), by joining threerestriction fragments from the following sources. The first fragment isan approximately 1649 base pair PvuI to SalI restriction fragment ofpSP64 (Promega). The second fragment is an approximately 1368 base pairPvuI to SalI restriction fragment of pSP65 (Promega). The third fragmentis the approximately 3400 base pair XhoI to XhoI fragment of plasmid437-47.1.

Homology Vector 535-70.3

The plasmid 535-70.3 was constructed for the purpose of inserting theMDV gB, and gA genes and the NDV F gene into HVT. The F gene wasinserted as a cassette into homology vector 456-17.22 at the HindIIIsite located between the MDV gA and gB genes (see Junction B, FIG. 10A).The F gene is under the control of the HCMV immediate early promoter andfollowed by the HSV-1 TK poly adenylation signal. The F gene wasinserted in the same transcriptional orientation as the US2 gene in theparental homology vector. The cassette may be constructed utilizingstandard recombinant DNA techniques (Maniatis et al, 1982 and Sambrooket al, 1989), by joining restriction fragments from the followingsources. The first fragment is an approximately 1191 base pair PstI toAvaII restriction sub-fragment of the HCMV genomic XbaI E fragment (D.R. Thomsen, et al., 1981). The second fragment is an approximately 1812base pair BamHI to PstI restriction fragment of the full length NDV FcDNA clone (B1 strain). The last fragment is an approximately 784 basepair SmaI to SmaI restriction sub-fragment of the HSV-1 BamHIrestriction fragment Q (McGeoch, et al., 1985).

Homology Vector 549-24.15

The plasmid 549-24.15 was constructed for the purpose of inserting theMDV gB, and gA genes and the NDV HN and F genes into HVT. The HN and Fgenes were inserted as a cassette into homolgy vector 456-17.22 at theHindIII site located between the MDV gA and gB genes (see Junction B,FIG. 10A). The HN and F genes are under the control of the PRV gpX andHCMV immediate early promoters respectively. The HN and F genes arefollowed by the PRV gX poly and HSV-1 TK adenylation signalsrespectively. The cassette may be constructed utilizing standardrecombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al,1989), by joining restriction fragments from the following sources. Thefirst fragment is an approximately 413 base pair SalI to BamHIrestriction sub-fragment of the PRV BamHI fragment #10 (Lomniczi, etal., 1984). The second fragment is an approximately 1811 base pair AvaIIto NaeI restriction fragment of the full length NDV HN cDNA clone (B1strain). The third fragment is an approximately 754 base pair NdeI toSalI restriction sub-fragment of the PRV BamHI restriction fragment #7(Lomniczi, et al., 1984). The fourth fragment is an approximately 1191base pair PstI to AvaII restriction sub-fragment of the HCMV genomicXbaI E fragment (D. R. Thomsen, et al., 1981). The fifth fragment is anapproximately 1812 base pair BamHI to PstI restriction fragment of thefull length NDV F cDNA clone (B1 strain). The last fragment is anapproximately 784 base pair SmaI to SmaI restriction sub-fragment of theHSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

Homology Vector 549-62.10

The plasmid 549-62.10 was constructed for the purpose of inserting theMDV gB, and gA genes and the NDV HN gene into HVT. The HN gene wasinserted as a cassette into homolgy vector 456-17.22 at the HindIII sitelocated between the MDV gA and gB genes (see Junction B, FIG. 10A). TheHN gene is under the control of the PRV gpX promoter and followed by thePRV gX poly adenylation signal. The HN gene was inserted in the sametranscriptional orientation as the US2 gene in the parental homologyvector. The cassette may be constructed utilizing standard recombinantDNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), byjoining restriction fragments from the following sources. The firstfragment is an approximately 413 base pair SalI to BamHI restrictionsub-fragment of the PRV BamHI fragment #10 (Lomniczi, et al., 1984) Thesecond fragment is an approximately 1811 base pair AvaII to NaeIrestriction fragment of the full length NDV HN cDNA clone (B1 strain).The last fragment is an approximately 754 base pair NdeI to SalIrestriction sub-fragment of the PRV BamHI restriction fragment #7(Lomniczi, et al., 1984).

Subgenomic Clone 550-60.6

Plasmid 550-60.6 was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 12,300 base pair region ofgenomic HVT DNA. It may be used in conjunction with other subgenomicclones according to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUSFROM OVERLAPPING SUBGENOMIC FRAGMENTS for the construction ofrecombinant HVT. This plasmid may be constructed utilizing standardrecombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al,1989), by joining two restriction fragments from the following sources.The first fragment is an approximately 4176 base pair EcoRV to BamHIrestriction fragment of pBR322. The second fragment is the approximately12,300 base pair sub-fragment of the BamHI #2 fragment of HVT(Buckmaster et al., 1988). This fragment was generated in the followingmanner. Plasmid 437-26.26 was linearized with HindIII and then resectedwith the ExoIII Mung Bean Deletion Kit (Stratagene). Samples from the 3and 4 minute reactions were combined and digested with BamHI resultingin a population of fragments containing the desired 12,300 base pairsub-fragment. This population was cloned into the pBR322 fragment andthe resulting clones were screened for the appropriate size andrestriction map. Fortuitously the resected sub-fragment that generatedclone 550-60.6 ended in the nucleotides GG which generated a secondBamHI site when ligated to the EcoRV site (ATCC) of pBR322. A bacterialstrain containing this plasmid has been deposited on Mar. 3, 1993pursuant to the Budapest Treaty on the International Deposit ofMicroorganisms for the Purposes of Patent Procedure with the PatentCulture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No.75429.

Homology Vectors 566-41.5

The plasmid 566-41.5 was constructed for the purpose of inserting theMDV gA, gB and gD genes into HVT. The MDV gD gene was inserted as aHindIII fragment into the homology vector 456-17.22 at the HindIII sitelocated between MDV gA and gB (see FIGS. 10A and 10B). The MDV gene wasinserted in the same transcriptional orientation as gA and gB in theparental homology vector. A detailed description of the HindIII fragmentcontaining the MDV gD gene is shown in FIGS. 11A and 11B. Note that aherpesvirus polyadenation signal was added to the gD gene cassette. Theinserted HindIII fragment may be constructed utilizing standardrecombinant DNA techniques (Maniatis et al, 1982 and Sambrook et al,1989), by joining restriction fragments from the following sources withthe synthetic DNA sequences indicated in FIGS. 11A and 11B. Fragment 1is an approximately 784 base pair SmaI to SmaI restriction sub-fragmentof the HSV-1 BamHI restriction fragment Q (McGeoch et al., 1988). Notethat this fragment is oriented such that the polyadenylation sequence(AATAAA) is located closest to junction B. Fragment 2 is anapproximately 2177 base pair SalI to NcoI sub-fragment of the MDV BglII4.2 KB genomic restriction fragment (Ross, et al., 1991).

Homology Vector 567-72.1D

The plasmid 567-72.1D was constructed for the purpose of inserting theMDV gB, gA, and gD genes and the infectious bronchitis virus (IBV)matrix and spike genes into HVT. The IBV genes were inserted as acassette into homolgy vector 566-41.5 at the unique NotI site locatedupstream of the MDV gD gene (see Junction C, FIG. 11B). The IBV spikeand matrix genes are under the control of the HCMV immediate early andPRV gpX promoters respectively. The IBV spike and matrix genes arefollowed by the HSV-1 TK and PRV gX poly adenylation signalsrespectively. The IBV genes were inserted in the same transcriptionalorientation as the US2 gene in the parental homology vector. Thecassette may be constructed utilizing standard recombinant DNAtechniques (Maniatis et al, 1982 and Sambrook et al, 1989), by joiningrestriction fragments from the following sources. The first fragment isan approximately 413 base pair SalI to BamHI restriction sub-fragment ofthe PRV BamHI fragment #10 (Lomniczi, et al., 1984) The second fragmentcontains amino acids 1 to 223 of the IBV matrix gene. The coding regionwas obtained from a cDNA clone of the Arkansas strain of IBV. The thirdfragment is an approximately 754 base pair NdeI to SalI restrictionsub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al.,1984). The fourth fragment is an approximately 1191 base pair PstI toAvaII restriction sub-fragment of the HCMV genomic XbaI E fragment (D.R. Thomsen, et al., 1981). The fifth fragment contains amino acids 4 to1162 of the IBV spike gene. The coding region was obtained from a cDNAclone of the Arkansas strain of IBV. The last fragment is anapproximately 784 base pair SmaI to SmaI restriction sub-fragment of theHSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

Homology Vector 603-57.F1

The plasmid 603-57.F1 was constructed for the purpose of inserting theIBDV VP2 gene into HVT. The IBDV VP2 gene was inserted as a cassetteinto homolgy vector 435-47.1 at the unique HindIII site. The VP2 gene isunder the control of the HCMV immediate early promoter and is followedby the HSV-1 TK poly adenylation signal. The VP2 gene was inserted inthe same transcriptional orientation as the US2 in the parental homologyvector. The cassette may be constructed utilizing standard recombinantDNA techniques (Maniatis et al, 1982 and Sambrook et al, 1989), byjoining restriction fragments from the following sources. The firstfragment is an approximately 1191 base pair PstI to AvaII restrictionsub-fragment of the HCMV genomic XbaI E fragment (D. R. Thomsen, et al.,1981). The second fragment is an approximately 1081 base pair BclI toBamHI restriction sub-fragment of the full length IBDV cDNA clone (seeSEQ ID NO:1). Note that the BclI site was introduced into the cDNA clonedirectly upstream of the VP2 initiator methionine by converting thesequence CGCAGC to TGATCA. The first and second fragments are orientedsuch that AvaII and BclI sites are contiguous. The third fragment is anapproximately 784 base pair SmaI to SmaI restriction sub-fragment of theHSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

Homology Vector 633-13.27

The plasmid 633-13.27 was constructed for the purpose of inserting theMDV gB, gA and gD genes and the NDV HN and F genes into HVT. The HN andF genes are under the control of the PRV gpX and HCMV immediate earlypromoters respectively. The HN and F genes are followed by the PRV gXpoly and HSV-1 TK adenylation signals respectively. All five genes wereinserted in the same transcriptional orientation as the US2 gene in theparental homology vector. The genes were inserted in the following orderMDV gA, NDV HN, NDV F,MDV gD, and MDV gB.

Homology Vector 634-29.16

The plasmid 634-29.16 was constructed for the purpose of inserting theILT virus gB and gD genes into HVT. The lacZ marker gene followed by theILT gB and gD genes inserted as a cassette into the homology vector172-29.31 at the unique XhoI site. The cassette may be constructedutilizing standard recombinant DNA techniques (Maniatis et al, 1982 andSambrook et al, 1989), by joining restriction fragments from thefollowing sources. The first fragment is an approximately 4229 base pairSalI to SalI restriction fragment derived from the lacZ marker genedescribed above and shown in FIGS. 7A and 7B. The second fragment is anapproximately 2060 base pair EcoRI to BclI restriction sub-fragment ofthe ILT KpnI genomic restriction fragment #8 (10.6 KB). The thirdfragment is an approximately 754 base pair NdeI to SalI restrictionsub-fragment of the PRV BamHI restriction fragment #7 (Lomniczi et al.,1984). Note that the second and third fragments are oriented such thatBclI and NdeI sites are contiguous. The fourth fragment is the 3.0 KBILT virus genomic EcoRI fragment containing the gB gene. All three genesare in the same transcriptional orientation as the UL43 gene.

Subgenomic Clone 415-09.BA1

Cosmid 415-09.Bμl was constructed for the purpose of generatingrecombinant HVT. It contains an approximately 29,500 base pair BamHI #1fragment of genomic HVT DNA. It was used in conjunction with othersubgenomic clones according to the PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS for the constructionof recombinant HVT. This cosmid was constructed by joining tworestriction fragments (Sambrook, et al., 1989) from the followingsources. The vector is an approximately 4430 base pair BamHI to BamHIrestriction fragment of pSY1005 derived from pHC79 (Bethesda ResearchLabs, Inc.) and pWE15 (Stratagene, Inc.). The first fragment is theapproximately 29,500 base pair BamHI #1 fragment of the HVT genome(Buckmaster et al., 1988).

Subgenomic Clone 672-01.A40

Cosmid 672-01.A40 was constructed for the purpose of generatingrecombinant HVT. It was isolated as a subclone of cosmid 407-32.1C1 (seeFIGS. 8 and 15). Cosmid 672-01.A40 contains an approximately 14,000 basepair NotI to AscI subfragment and an approximately 1300 base pair AscIto BamHI subfragment of cosmid 407-32.1C1. The cosmid was constructed byjoining restriction fragments (Sambrook, et al., 1989) from thefollowing sources. The vector is an approximately 2700 base pair NotI toBamHI fragment constructed from pNEB193 (New England Biolabs, Inc.)which contains a NotI linker inserted into the SmaI site. Fragment 1 isan approximately 15,300 base pair region of genomic HVT DNA. This regionincludes BamHI fragments 11 and 7, and approximately 1250 base paris offragment 13. It was used in conjunction with other subgenomic clonesaccording to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROMOVERLAPPING SUBGENOMIC FRAGMENTS for the construction of recombinantHVT.

Subgenomic Clone 654-45.1.

Plasmid 654-45.1 was constructed for the purpose of generatingrecombinant HVT. It was isolated as an AscI subclone of cosmid407-32.1C1 (see FIGS. 8 and 15). The cosmid was constructed by joiningrestriction fragments (Sambrook, et al., 1989) from the followingsources. The vector is an approximately 2000 base pair AscI fragmentconstructed from a 2000 base pair AatII to PvuII fragment of pNEB 193(New England Bilabs, Inc.) blunt ended with Klenow DNA polymerase andAscI linkers inserted. Fragment 1 is an approximately 8600 base pairAscI to AscI fragment of genomic HVT DNA. This region includes BamHIfragments 10 and 21, and approximately 1100 base pairs of fragment 6 andapproximately 1300 base pairs of fragment 7. The XhoI site (Nucleotide#1339-1344; SEQ ID NO. 48) has been converted to a unique PacI siteusing synthetic DNA linkers. The PacI site was used in insertion andexpression of foreign genes in HVT. (See FIG. 13A). It was used inconjunction with other subgenomic clones according to the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTSfor the construction of recombinant HVT.

Subgenomic Clone 686-63.A1

Plasmid 686-63.A1 was constructed for the purpose of generatingrecombinant HVT. It was isolated as an AscI subclone of cosmid407-32.1C1 (see FIG. 8, 15). The cosmid was constructed by joiningrestriction fragments (Sambrooks, et al., 1989) from the followingsources. The vector is an approximately 2000 base pair AscI fragmentconstructed from a 2000 base pair AatII to PvuII fragment of pNEB193(New England Biolabs, Inc.) blunt ended with Klenow DNA polymerase andAscI linkers inserted. Fragment 1 is an approximately 8600 base pairAscI to AscI fragment of genomic HVT DNa. This region includes BamHIfragments 10 and 21, and approximately 1100 base pairs of fragment 6 andapproximately 1300 base pairs of fragment 7. The XhoI site (Nucleotide#1339-1344; SEQ ID NO. 48) has been converted to a unique NotI siteusing synthetic DNA linkers. The NotI site was used for the insertionand expression of foreign genes in HVT. (See FIG. 13B). It was used inconjunction with other subgenomic clones according to the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTSfor the construction of recombinant HVT.

Subgenomic Clone 672-07.C40

Cosmid 672-07.C40 was constructed for the purpose of generatingrecombinant HVT. It was isolated as a subclone of cosmid 407-32.1C1 (seeFIGS. 8 and 15). Cosmid 672-07.C40 contains an approximately 1100 basepair BamHI to AscI subfragment and an approximately 13,000 base pairAscI to NotI subfragment of cosmid 407-32.1C1. The cosmid wasconstructed by joining restriction fragments (Sambrook, et al., 1989)from the following sources. The vector is an approximately 2700 basepair NotI to BamHI fragment constructed from pNEB193 (New EnglandBiolabs, Inc.) which contains a NotI linker inserted into the SmaI site.Fragment 1 is an approximately 14,100 base pair region of genomic HVTDNA. This region includes BamHI fragments 6 and 18, and an approximately2600 base pair BamHI to NotI fragment within BamHI fragment #1. It wasused in conjunction with other subgenomic clones according to thePROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPINGSUBGENOMIC FRAGMENTS for the construction of recombinant HVT.

Subgenomic Clone 706-57.A3

Plasmid 706-57.A3 was constructed for the purpose of generatingrecombinant HVT. Plasmid 706-57.A3 contains the IBDV VP2 gene insertedinto the PacI site of plasmid 654-45.1. The IBDV VP2 gene uses the IBRVVP8 promoter and ILTV US3 polyadenylation signal. The cosmid wasconstructed utilizing standard recombinant DNA techniques (Sambrook, etal., 1989). The first fragment is a 208 base pair HindIII to BamHIfragment coding for the IBRV VP8 promoter (Carpenter, et al., 1991)).The second fragment is an approximately 1626 base pair fragment codingfor the IBDV VP2 gene derived by reverse transcription and polymerasechain reaction (Sambrook, et al., 1989) of IBDV standard challengestrain (USDA) genomic RNA (Kibenge, et al., 1990). The antisense primerused for reverse transcription and PCR was5'-CTGGTTCGGCCCATGATCAGATGACAAACCTGCAAGATC-3' (SEQ ID NO. 53). The senseprimer used for PCR was 5'-CTGGTTCGGCCCATGATCAGATGACAAACCTGCAAGATC-3'(SEQ ID NO. 54). The DNA fragment generated by PCR was cloned into thePCR-Direct™ vector (Clontech Laboratories, Inc. Pali Alto, Calif.). TheIBDV VP2 fragment was subcloned next tot he VP8 promoter using BclIsites generated by the PCR primers. The DNA sequence at this junctionadds amino acids methionine, aspartate and glutamine before the antiveinitiator methionine of VP2. The DNA fragment contains the codingsequence from amino acid 1 to amino acid 536 of the IBDV polyprotein(SEQ ID NO: 2) which includes the entire coding sequence of the VP2protein. The third fragment is an approximately 494 base pair fragmentcoding for the ILTV US3 polyadenylation signal.

Subgenomic Clone 711-92.1A

Plasmid 711-92.1A was constructed for the purpose of generatingrecombinant HVT. Plasmid 711-92.1A contains the ILTV gD and gI genesinserted into the PacI site of plasmid 654-45.1. The ILTV gD and gIgenes use their respective endogenous ILTV promoters and single sharedendogenous polyadenylation signal. The plasmid was constructed utilizingstandard recombinant DNA techniques (Sambrook, et al., 1989). The firstfragment is an approximately 3556 base pair SalI to HindIII restrictionsubfragment of the ILTV Asp718I genomic fragment #8 (10.6 kb).

Subgenomic Clone 717-38.12

Plasmid 717-38.12 was constructed for the purpose of generatingrecombinant HVT. Plasmid 717-38.12 contains the NDV HN and F genesinserted into the PacI site of plasmid 654-45.1. The NDV HN gene usesthe PRV gX promoter and the PRV gX polyadenylation signal. The NDV Fgene uses the HCMV immediate early promoter and the HSV TKpolyadenylation signal. The plamid was constructed utilizing standardrecombinant DNA techniques (Sambrook, et al., 1989). The first fragmentis an approximately 413 base pair SalI to BamHI restriction subfragmentof the PRV BamHI fragment #10 (Lomniczi, et al., 1984). The secondfragment is an approximately 1811 base pair AvaII to NaeI restrictionfragment of the full length NDV HN cDNA clone (B1 strain). The thirdfragment is an approximately 754 base pair NdeI to SalI restrictionsubfragment of the PRV BamHI restriction fragment #7 (Lomniczi, et al.,1984). The fourth fragment is an approximately 1191 base pair PstI toAvaII restriction subfragment of the HCMV genomic XbaI E fragment (D. R.Thomsen, et al., 1981). The fifth fragment is an approximately 1812 basepair BamHI to PstI restriction fragment of the full length NDV F cDNAclone (B1 strain; SEQ ID NO: 12). The sixth fragment is an approximately784 base pair SmaI to SmaI restriction subfragment of the HSV-1 BamHIrestriction fragment Q (McGeoch, et al., 1985).

Subgenomic Clone 721-38.1J

Cosmid 721-38.1J was constructed for the purpose of inserting the MDVgA, gD, and gB genes into the unique short of HVT and for the purpose ofgenerating recombinant HVT. Cosmid 721-38.1J contains the MDV gA, gD andgB genes inserted into a StuI site in the HVT US2 gene converted to aunique HindIII site within the BamHI #1 fragment of the unique shortregion of HVT. This region of the HVT BamHI #1 fragment containing theMDV genes was derived from S-HVT-062. Cosmid 721-38.1J was constructedby a partial restriction digest with BamHI of S-HVT-062 DNA andisolation of an approximately 39,300 base pair fragment. The cosmid wasconstructed utilizing standard recombinant DNA techniques (Sambrook, etal., 1989) by joining restriction fragments from the following sources.The vector is an approximately 8200 base pair BamHI fragment from cosmidvector pWE15. The first fragment is an approximately 900 base pair BamHIfragment from the repeat region of the HVT genome. The second fragmentis an approximately 15,500 base pair BamHI to StuI subfragment of BamHI#1 of HVT. The third fragment is an approximately 8400 base paircassette containing the MDV gA, gD, and gB genes (see FIGS. 10 and 11).The fourth fragment is an approximately 14,500 base pair HindIII toBamHI subfragment of the BamHI #1 of HVT.

Subgenomic Clone 722-60.E2

Cosmid 722-60.E2 was constructed for the purpose of inserting the MDVgA, gD, and gB genes and the NDV HN and F genes into the unique short ofHVT and for the purpose of generating recombinant HVT. Cosmid 722-60.E2contains the MDV gA, gD and gB genes and the NDV HN and F genes insertedinto a StuI site in the HVT US2 gene converted to a unique HindIII sitewithin the BamHI #1 fragment of the unique short region of HVT. All fivegenes were inserted in the same transcriptional orientation as the HVTUS2 gene. This region of the HVT BamHI #1 fragment containing the MDVand NDV genes was derived from S-HVT-106. Cosmid 722-60.E2 wasconstructed by a partial restriction digest with BamHI of S-HVT-106 andisolation of an approximately 46,300 base pair fragment. The cosmid wasconstructed utilizing standard recombinant DNA techniques (Sambrook, etal., 1989) by joining restriction fragments from the following sources.The vector is an approximately 6100 base pair BamHI fragment from cosmidvector pSY1626 derived from pHC79 (Bethesda Research Labs, Inc.) andpWE15 (Strategene, Inc.). The first fragment is an approximately 900base pair BamHI fragment from the repeat region of the HVT genome. Thesecond fragment is an approximately 15,500 base pair BamHI to StuIsubfragment of BamHI #1 of HVT. The third fragment is an approximately15,400 base pair cassette containing the MDV gA gene, (FIGS. 10A and10B, SEQ ID NO: 8), the PRV gX promoter (Lomniczi et al., 1984), the NDVHN gene (SEQ ID NO: 10), the PRV gX polyadenylation site (Lomniczi etal., 1984), the HCMV immediate early promoter (D. R. Thomsen, et al.,1981), the NDV F gene (SEQ ID NO: 12), the HSV TK polyadenylation site(McGeoch, et al., 1985), the MDV gD gene (FIGS. 11A and 11B), theapproximately 450 base pair ILTV US3 polyadenylation site, and the MDVgB gene (FIGS. 10A and 10B). The fourth fragment is an approximately14,500 base pair StuI to BamHI subfragment of the BamHI #1 of HVT.

Subgenomic Clone 729-37.1

Plasmid 729-37.1 was constructed for the purpose of generatingrecombinant HVT. Plasmid 729-37.1 contains the ILTV gD and gB genesinserted into the NotI site of plasmid 686-63.A1. The ILTV gD and gBgenes use their respective endogenous ILTV promoters, and the ILTV gDand gB gene are each followed by a PRV gX polyadenylation signals. TheILTV gD and gB gene cassette was constructed utilizing standardrecombinant DNA techniques (Sambrook, et al., 1989). The first fragmentis an approximately 2052 base pair SalI to XbaI restriction subfragmentof the ILTV Asp718I genomic fragment #8 (10.6 kb). The second fragmentis an approximately 572 base pair XbaI to Asp718I restrictionsubfragment of the PRV BamHI restriction fragment #7 (Lomniczi et al.,1984). The third fragment is an approximately 3059 base pair EcoRI toEcoRI restriction fragment of ILTV genomic DNA. The fourth fragment isan approximately 222 base pair EcoRI to SalI restriction subfragment ofthe PRV BamHI restriction fragment #7 (Lomniczi et al., 1984).

Subgenomic Clone 739-27.16

Cosmid 739-27.16 was constructed for the purpose of constructingachimeric HVT/MDV virus containing the HVT genes of the unique longregion and the MDV type 1 genes of the unique short region. Cosmid739-27.16 contains the complete unique short region of MDV type 1. Thisregion contains the entire SmaI B fragment and two SmaI K fragments.Cosmid 739-27.16 was constructed by a partial restriction digest withSmaI of MDV DNA and isolation of an approximately 29,000 to 33,000 basepair fragment. The cosmid was constructed utilizing standard recombinantDNA techniques (Sambrook, et al., 1989) by joining restriction fragmentsfrom the following sources. The vector is an approximately 8200 basepair BamHI fragment (made blunt-ended with Lenow DNa polymerase) fromcosmid vector pWE15. The first fragment is an approximately 4050 basepair SmaI K fragment from the short internal repeat region of the MDVgenome. The second fragment is an approximately 21,000 base pairfragment SmaI B of MDV. The third fragment is an approximately 3,650base pair SmaI K fragment from the short terminal repeat region of theMDV genome (Fukuchi, et al., 1984, 1985).

Subgenomic Clone 751-87.A8

Plasmid 751-87.A8 was constructed for the purpose of generatingrecombinant HVT. Plasmid 751-87.A8 contains the chicken myelomonocyticgrowth factor (cGMF) gene inserted into the PacI site of plasmid654-45.1. The cMGF gene uses the HCMV immediate early promoter and HSV-1TK polyadenylation signal. The cosmid was constructed utilizing standardrecombinant DNA techniques (Sambrook, et al., 1989). The followingfragments were inserted into the PacI site of HVT subgenomic clone654-45.1. The first fragment is an approximately 1191 base pair PstI toAvaII restriction subfragment of the HCMV genomic XbaI E fragment (D. R.Thomsen, et al., 1981). The second fragment is an approximately 640 basepair fragment coding for the cMGF gene (58) derived by reversetranscription and polymerase chain reaction (PCR) (Sambrook, et al.,1989) of RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEENCELLS. The antisense primer used for reverse transcription and PCR was5'-CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3' (SEQ ID NO: 57). The senseprimer used for PCR was 5'-GAGCGGATCCTGCAGGAGGAGACACAGAGCTG-3' (SEQ IDNO: 58). The cMGF fragment was subcloned next to the HCMV IE promoterusing BamHI sites generated by the PCR primers. The DNA fragmentcontains the coding sequence from amino acid 1 to amino acid 201 of thecMGF protein (58) which includes a 23 amino acid leader sequence at theamino terminus and 178 amino acids of the mature cMGF protein. The thirdfragment is an approximately 784 base pair SmaI to SmaI restrictionsubfragment of the HSV-1 BamHI restriction fragment Q (McGeoch, et al.,1985).

Subgenomic Clone 761-07.A1

Plasmid 761-07.A1 was constructed for the purpose of generatingrecombinant HVT. Plasmid 761-07.A1 contains the chicken interferon geneinserted into the PacI site of plasmid 654-45.1. The chicken interferongene uses the HCMV immediate early promoter and HSV-1 TK polyadenylationsignal. The cosmid was constructed utilizing standard recombinant DNAtechniques (Sambrook, et al., 1989). The following fragments wereinserted into the PacI site of HVT subgenomic clone 654-45.1. The firstfragment is an approximately 1191 base pair PstI to AvaII restrictionsubfragment of the HCMV genomic XbaI E fragment (D. R. Thomsen, et al.,1981). The second fragment is an approximately 577 base pair fragmentcoding for the chicken interferon gene (59) derived by reversetranscription and polymerase chain reaction (PCR) (Sambrook, et al.,1989) of RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEENCELLS. The antisense primer used for reverse transcription and PCR was5'-TGTAGAGATCTGGCTAAGTGCGCGTGTTGCCTG-3' (SEQ ID NO: 59). The senseprimer used for PCR was 5'-TGTACAGATCTCACCATGGCTGTGCCTGCAAGC-3' (SEQ IDNO: 60). The chicken interferon gene fragment was subcloned next to theHCMV IE promoter using BglII sites generated by the PCR primers. The DNAfragment contains the coding sequence from amino acid 1 to amino acid193 of the chicken interferon protein (59) which includes a 31 aminoacid signal sequence at the amino terminus and 162 amino acids of themature protein encoding chicken interferon. The third fragment is anapproximately 784 base pair SmaI to SmaI restriction subfragment of theHSV-1 BamHI restriction fragment Q (McGeoch, et al., 1985).

EXAMPLE 1

S-HVT-001

S-HVT-001 is a herpesvirus of turkeys (HVT) that contains the E. coliβ-galactosidase gene inserted into the unique long region of the HVTgenome. The restriction enzyme map of HVT has been published (T.Igarashi, et al., 1985). This information was used as a starting pointto engineer the insertion of foreign genes into HVT. The BamHIrestriction map of HVT is shown in FIG. 1A. From this data, severaldifferent regions of HVT DNA into which insertions of foreign genescould be made were targeted. The foreign gene chosen for insertion wasthe E. coli β-galactosidase (lacZ) gene , which was used in PRV. Thepromoter was the PRV gpX promoter. The lacZ gene was inserted into theunique long region of HVT, specifically into the XhoI site in the BamHI#16 (3329 bp) fragment, and was shown to be expressed in an HVTrecombinant by the formation of blue plaques using the substrateBluogal™ (Bethesda Research Labs). Similarly, the lacZ gene has beeninserted into the SalI site in the repeat region contained within theBamHI #19 (900 bp) fragment.

These experiments show that HVT is amenable to the procedures describedwithin this application for the insertion and expression of foreigngenes in herpesviruses. In particular, two sites for insertion offoreign DNA have been identified (FIGS. 1B and 1C).

EXAMPLE 2

S-HVT-003

S-HVT-003 is a herpesvirus of turkeys (HVT) that contains the E. coliβ-galactosidase (lacZ) gene and the infectious bursal disease virus(IBDV) strain S40747 large segment of RNA (as a cDNA copy) (SEQ IDNO: 1) inserted into the unique long region of the HVT genome. This IBDVDNA contains one open reading frame that encodes three proteins(5'VP2-VP4-VP3 3') (SEQ ID NO: 2), two of which are antigens to provideprotection against IBDV infections of chickens. Expression of the genesfor both β-galactosidase and the IBDV polyprotein are under the controlof the pseudorabies virus (PRV) gpX gene promoter. S-HVT-003 was made byhomologous recombination. S-HVT-003 was deposited on Jul. 21, 1987pursuant to the Budapest Treaty on the International Deposit ofMicroorganism for Purposes of Patent Procedure with the Patent CultureDepository of the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR 2178.

The IBDV genes were cloned by the cDNA CLONING PROCEDURE. Clonesrepresenting the genome of IBDV were screened by SOUTHERN BLOTTING OFDNA procedure against blots containing authentic IBDV RNA. Positiveclones were then characterized by restriction mapping to identify groupsof clones. Two such clones were identified, that together were found torepresent the entire coding region of the IBDV large segment of RNA (3.3kb dsRNA). One cDNA clone (2-84) contained an approximately 2500 basepair fragment representing the first half of the IBDV gene. The secondclone (2-40) contained an approximately 2000 base pair fragmentrepresenting the distal half of the IBDV gene. Plasmid 2-84/2-40,representing the entire IBDV gene, was constructed by joining clone 2-84and 2-40 at a unique PvuII site present in the overlapping sequences.The IBDV genome can be obtained from plasmid 2-84/2-40 as anapproximately 3400 base pair SmaI to HpaI fragment. Confirmation of thenature of the proteins encoded by the IBDV gene was obtained byexpressing the clone (2-84/2-40) in E. coli and detecting VP3 antigenusing antiserum made against purified IBDV capsid proteins on Westernblots. The cDNA of the IBDV large segment of RNA encoding the IBDVantigens show one open reading frame that will henceforth be referred toas the IBDV gene. The sequence of an Australian IBDV strain has beenpublished which bears close homology to applicants' sequence (Hudson etal,1986). Comparison of the amino acid differences between the twoviruses revealed 29 amino acid changes within the 1012 amino acid codingregion. There were only 3 amino acid differences deduced for VP4 andonly 8 in VP3. In contrast, VP2 contained 18 amino acid changes, 14 ofwhich were clustered between amino acids 139 to 332.

For insertion into the genome of HVT, the coding region for the IBDVgene was cloned between the PRV gpX promoter and the HSV TK poly-Asignal sequence, creating plasmid 191-23. To aid in the identificationof HVT recombinants made by homologous recombination containing the IBDVgene, the gpX promoted IBDV fragment from plasmid 191-23 was insertedbehind (in tandem to) a lacZ gene controlled by a gpX promoter. Theresultant plasmid, 191-47, contains the E.coli lacZ gene and the IBDVgene under the control of individual PRV gpX promoters. In constructingplasmid 191-47, various DNA fragments were joined by recombinant DNAtechniques using either naturally occurring restriction sites orsynthetic linker DNA. Details concerning the construction of these genescontained in plasmid 191-47 can be seen in FIGS. 2A, 2B, 2C and 2D.

The first segment of DNA (Segment 1, FIG. 2A) contains the gpX promoterregion including the residues encoding the first seven amino acids ofthe gpX gene, and was derived from a subclone of the PRV BamHI #10fragment as an approximately 800 base pair SalI to BamHI fragment. Thesecond segment of DNA (Segment 2, FIG. 2A) contains the E. coliβ-galactosidase coding region from amino acid 10 to amino acid 1024 andwas derived from the plasmid pJF751 (obtained from Jim Hoch, ScrippsClinic and Research Foundation) as an approximately 3300 base pair BamHIto BalI fragment followed by an approximately 40 base pair Ava I to SmaI fragment. The third segment of DNA (Segment 3, FIG. 2A) contains thegpX poly A signal sequence and was derived from a subclone of the PRVBamHI #7 fragment as an approximately 700 base pair NdeI to StuIfragment. Segment three was joined to segment two by ligating the NdeIend which had been filled in according to the POLYMERASE FILL-INREACTION, to the SmaI site. The fourth segment of DNA (Segment 4, FIG.2A) contains the gpX promoter (TATA box and cap site) and was derivedfrom a subclone of the PRV BamHI #10 fragment as an approximately 330base pair NaeI to AluI fragment. Additionally, segment four containsapproximately 36 base pairs of HSV TK 5'untranslated leader sequence asa PstI to BglII fragment in which the PstI site has been joined to theAluI site through the use of a synthetic DNA linker (McKnight andKingbury, 1982). DNA segments four through six were inserted as a unitinto the unique Kpn I site of segment three which is located 3' of thegpX poly A signal sequence. The fifth segment of DNA (Segment 5, FIG.2A) contains the entire coding region of the IBDV large segment of RNA(cDNA clone) as an approximately 3400 base pair SmaI to HpaI fragment.The SmaI site of segment five was fused to the BglII site of segmentfour which had been filled in according to the POLYMERASE FILL INREACTION. Expression of the IBDV gene (5'VP2-VP4-VP3 3') is under thecontrol of the gpX promoter (segment 4), but utilizes its own naturalstart and stop codons. The sixth segment of DNA (Segment 6, FIG. 2A)contains the HSV TK poly-A signal sequence as an approximately 800 basepair SmaI fragment (obtained from Bernard Roizman, Univ. of Chicago).The HpaI site of segment five was fused to the SmaI site of segment sixthrough the use of a synthetic DNA linker.

In summary, the construct used to create S-HVT-003 (plasmid 191-47)contains (5' to 3') the PRV promoter, the gpX TATA box, the gpX capsite, the first seven amino acids of gpX, the E. coli β-galactosidase(lacZ) gene, the PRV poly-A signal sequence, the PRV gpX promoter, thegpX TATA box, the gpX cap site, a fusion within the gpX untranslated 5'leader to the IBDV gene, IBDV start codon, a fusion within the IBDVuntranslated 3' end to HSV TK untranslated 3' end, and the TK poly-Asignal sequence. The cassette containing these genes was engineered suchthat it was flanked by two EcoRI restriction endonuclease sites. As aresult, an approximately 9100 base pair fragment containing both lacZgene and the IBDV gene can be obtained by digestion with EcoRI.Henceforth, the 9161 base pair EcoRI fragment will be referred to as theIBDV/lacZ cassette. The following procedures were used to constructS-HVT-003 by homologous recombination. The IBDV/lacZ cassette wasinserted into the unique XhoI site present within a subclone of the HVTBamHI #16 fragment. To achieve this, the XhoI site was first changed toan EcoRI site through the use of an EcoRI linker. This site hadpreviously been shown to be nonessential in HVT by the insertion of lacZ(S-HVT-001). It was also shown that the flanking homology regions inBamHI #16 were efficient in homologous recombination. Shown in FIGS. 3Aand 3B, the genomic location of the BamHI #16 fragment maps within theunique long region of HVT. The BamHI #16 fragment is approximately 3329base pairs in length (SEQ ID NOs: 3, 4, 5, 6, and 7). HVT DNA wasprepared by the PREPARATION OF HERPESVIRUS DNA procedure.Cotransfections of HVT DNA and plasmid DNA into primary chick embryofibroblast (CEF) cells were done according to the DNA TRANSFECTION FORGENERATING RECOMBINANT HERPESVIRUS. The recombinant virus resulting fromthe cotransfection stock was purified by three successive rounds ofplaque purification using the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUSprocedure. When 100% of the plaques were blue, the DNA was analyzed forthe presence of the IBDV gene by the SOUTHERN BLOTTING OF DNA procedure.Southern blots, probing EcoRI digested S-HVT-003 DNA with an IBDVspecific nick translated probe (plasmid 2-84/2-40), confirmed thepresence of the 9100 base pair EcoRI fragment. This result confirmedthat S-HVT-003 contained both the lacZ gene and the IBDV geneincorporated into its genome. Additional Southern blots, using a probespecific for BamHI #16, confirmed that the homologous recombinationoccurred at the appropriate position in BamHI #16 and that no deletionswere created. No differences in the growth of S-HVT-003 compared to wildtype virus (S-HVT-000) were observed in vitro.

Expression of IBDV specific proteins from S-HVT-003 were assayed invitro using the WESTERN BLOTTING PROCEDURE. Cellular lysates wereprepared as described in PREPARATION OF HERPESVIRUS CELL LYSATES.Briefly, the proteins contained in the cellular lysates of S-HVT-003were separated by polyacrylamide gel electrophoresis, transferred tonitrocellulose, and probed with either an antiserum made againstdenatured purified IBDV capsid proteins or antiserum made against asynthetic peptide corresponding to a predicted imuno dominant region ofthe IBDV 40 kd (VP2) capsid protein. The filters were washed and treatedwith [¹²⁵ I] protein A to detect the position of the bound antibodies.FIG. 4 shows the results obtained using the antiserum made againstdenatured purified IBDV capsid proteins, which have been shown by theapplicants to react primarily with VP3 (32 kd protein). As seen,S-HVT-003 produces a protein which is immunologically indistinguishablefrom the authentic VP3 protein from intact IBDV virions. Moreover, thepolyprotein appears to be processed correctly, producing a VP3 speciesthat comigrates with the authentic VP3 protein. Recent evidence using anAustralian IBDV stain indicates that VP4 is involved in the processingof the precursor polyprotein into mature VP2 and VP3 protein species(Jagadish, et al., 1988). FIG. 5 shows the results obtained using arabbit antiserum raised against a synthetic peptide that is homologousto a 14 amino acid region of the IBDV VP2 (40 kd) capsid protein. Asseen, S-HVT-003 produces a protein that is immunologicallyindistinguishable from the authentic viral VP2 protein. In addition, theVP2 protein produced from S-HVT-003 comigrates with the 40 kd species ofVP2 isolated from intact IBDV virions. This species represents a majorcomponent of infectious (complete) viral particles.

In summary, analysis of the expression of IBDV specific proteins fromS-HVT-003 has shown that the polyprotein is processed in CEF cellculture, producing proteins of the appropriate size that react toimmunological reagents specific for either VP2 or VP3 proteins onWestern blots.

The following set of experiments was carried out in chickens to analyzethe in vivo expression of the IBDV genes contained within S-HVT-003 asdetermined by seroconversion data, serum neutralization results, andprotection from IBDV challenge.

The first experiment was designed to show the seroconversion of chickensto IBDV upon being vaccinated with S-HVT-003. Eleven 11-week-oldchickens, seronegative to HVT and IBDV were obtained from SPAFAS Inc.Six birds were vaccinated subcutaneously in the abdominal region with0.5 ml of a cellular suspension of CEF cells containing S-HVT-003(40,000 PFU/ml). Serum samples were obtained every seven days for eightweeks for all birds in this study. On day 28 (4th week), three of thesebirds received a boost of S-HVT-003, while the other three birdsreceived 0.5 ml of an inactivated IBDV vaccine inoculated subcutaneouslyin the cervical region. Three additional birds were given only theinactivated vaccine on day 28. Two birds served as contact controls andreceived no vaccinations. On day 56, all birds were sacrificed andnecropsied. Table 1 show the results of the serum neutralization assayagainst IBDV. No detectable SN activity was observed in the birds givenonly S-HVT-003. Additionally, only one of the three birds that weregiven only the inactivated vaccine demonstrated low but detectable SNactivity. SN titers were also detected in one of the three birds thatreceived the S-HVT-003 followed by the inactivated IBDV vaccine boost;these titers were at a much higher level than with the inactivated IBDVvaccine alone. These results suggest that S-HVT-003 is priming thechicken for a secondary response against IBDV. In vitro analysis of theserum samples by WESTERN BLOTTING confirmed the seroconversion of thechickens to IBDV upon vaccination with S-HVT-003 both prior to and afterboosts administered on day 28.

                  TABLE 1                                                         ______________________________________                                                     DAY                                                              Vaccine Group                                                                           Bird No. 28     31  35   38   42    49                              ______________________________________                                        HVT-003   265      <2     <2  <2   <2   <2    <2                                HVT-003 266 <2 <2 <2 <2 <2 <2                                                  267 <2 <2 <2 <2 <2 <2                                                        HVT-003 260 <2 <2 <2 <2 <2 <2                                                 IBDV.sup.a 264 <2 <2 <2 1:64 1:256 1:512                                       269 <2 <2 <2 <2 <2 <2                                                        C 261 <2 <2 <2 <2 <2 <2                                                       IBDV.sup.a 262 <2 <2 <2 <2 1:4 1:4                                             263 <2 <2 <2 <2 <2 <2                                                        C 270 <2 <2 <2 <2 <2 <2                                                        271 <2 <2 <2 <2 <2 <2                                                      ______________________________________                                         .sup.a Commercial                                                        

In the second experiment, twenty five 1-day old SPF chicks werevaccinated with S-HVT-003 (20 with 0.2 ml subcutaneously and 5 bybilateral eyedrop). Twenty chicks were kept as controls. On days fourand seven postinfection, five vaccinates and two control birds werebled, sacrificed and their spleens removed for virus isolation. Spleencell suspensions were made by standard method, and ˜1×10⁶ cells in 3 mlof chick embryo fibroblast (CEF) growth media were inoculated directlyonto secondary cells. Cultures were incubated for 6-7 days and thenscored for cytopathic effects (CPE) as determined by observing cellmorphology. The cultures were passed a second time, and again scored forCPE. The results are shown in Table 2. All nonvaccinated control birdsremained negative for HVT for both day 4 and 7 spleen cell isolations.Four out of the five birds vaccinated with S-HVT-003 were positive forHVT at day 4 for both the first and second passages. One bird did notproduce virus, this may represent a vaccination failure. Five out offive birds were positive for HVT on day 7 at both passage one and two.Overall, the vector recovery experiment demonstrates that S-HVT-003replicates as well as wild type HVT virus in vivo and that insertion ofthe IBDV/lacZ cassette into the XhoI site of BamHI #16 does not resultin detectable attenuation of virus. Subsequent experiments examining therecovered virus by the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUSprocedure confirmed the in vivo stability of S-HVT-003, by demonstratingβ-galactosidase expression in 100% of the viruses.

                  TABLE 2                                                         ______________________________________                                        Harvest Date                                                                             Day 4        Day 7                                                 Sample     P1    P2         P1  P2                                            ______________________________________                                        N1         -     -                                                              N2 - -                                                                        N3   - -                                                                      N4   - -                                                                      T1 - -                                                                        T2 2+ 2+                                                                      T3 2+ 2+                                                                      T4 + 4+                                                                       T5 3+ 3+                                                                      T6   2+ contaminated                                                          T7   + 5+                                                                     T8   + 5+                                                                     T8   + 5+                                                                     T9   + 5+                                                                     T10   + 5+                                                                  ______________________________________                                         N = control, T = vaccinated                                                   CPE ranged from negative (-) to 5                                        

At days 0, 4, 7, 14, 21, and 27 postinfection, blood samples wereobtained from the rest of the chickens for determining serum ELISAtiters against IBDV and HVT antigens as well as for virus neutralizingtests against IBDV. Additionally, at 21 days postinfection five controland fourteen vaccinated chicks were challenged with virulent IBDV bybi-lateral eyedrop (10³.8 EID₅₀). All birds were sacrificed 6-days postchallenge and bursa to body weight ratios were calculated. A summary ofthe results is shown in tables 3 and 4, respectively. As presented inTable 3, no antibodies were detected against HVT antigens by ELISA priorto 21-27 days post vaccination. In chickens, the immune response duringthe first two weeks post hatch is both immature and parentallysuppressed, and therefore these results are not totally unexpected. Incontrast, IBDV ELISA's were negative up to day 21 post-vaccination, andwere only detectable after challenge on day 27. The ELISA levels seen onday 27 post-vaccination indicate a primary response to IBDV. Table 4comparing the Bursa-to-Body weight ratios for challenged controls andvaccinated/challenged groups show no significant differences.Vaccination with S-HVT-003 under these conditions did not preventinfection of the vaccinated birds by IBDV challenge, as indicated by thedeath of four vaccinated birds following challenge.

                  TABLE 3                                                         ______________________________________                                                 ELISA         VN                                                     Sample Group                                                                             HVT         IBDV    IBDV                                           ______________________________________                                        C-0 (n = 3)                                                                              0           0       <100                                             C-4 (n = 2) 0 0 nd                                                            T-4 (n = 5) 0 0 nd                                                            C-7 (n = 2) 0 0 <100                                                          T-7 (n = 5) 0 0 <100                                                          C-14 (n = 5) 0 0 nd                                                           T-14 (n = 14) 0 0 <100                                                        C-21 (n = 5) 0 0 nd                                                           T-21 (n = 14) 1 0 <100                                                        C-27 (n = 5) 0 0 nd                                                           CC-27 (n = 5) 0 5 nd                                                          CT-27 (n = 10) 3.2 2 nd                                                     ______________________________________                                         C = control                                                                   T = vaccinated                                                                CC = challenged control                                                       CT = Challenged & vaccinated.                                                 ELISA titers are GMTs and they range from 0-9.                           

                  TABLE 4                                                         ______________________________________                                        Sample Group                                                                             Body wt.     Bursa wt.                                                                              BBR                                          ______________________________________                                        Control (n = 5)                                                                          258.8        1.5088   0.0058                                         Challenge 209   0.6502 0.0031                                                 Control (n = 5)                                                               Challenge 215.5 0.5944 0.0027                                                 Treated (n = 10)                                                            ______________________________________                                    

Values are mean values. Body weights are different in control groupbecause challenged birds did not feed well. Four challenged-treatedbirds died.

A third experiment was conducted repeating Experiment 2 but usingimmunologically responsive chicks (3 weeks of age). Six three week oldSPF leghorn chickens were vaccinated intraperitoneally with 0.2 ml ofS-HVT-003 (one drop in each eye). Serum samples were obtained everyseven days for six-weeks and the birds were challenged with the virulentUSDA standard challenge IBDV virus on day 43 post-vaccination. Six dayspost challenge, the control, vaccinated-challenged, and challengedgroups were sacrificed and bursas were harvested for probing withanti-IBDV monoclonal antibodies (MAB) (provided by Dr. David Snyder,Virginia-Maryland Regional College of Veterinary Medicine). Bursalhomogenates were prepared by mixing 1 ml of 0.5% NP40 with one bursa.Bursa were then ground and briefly sonicated. Supernatants from thehomogenates were reacted with the R63 MAB which had been affixed to96-well Elisa plates via a protein A linkage. After incubation, a biotinlabeled preparation of the R63 MAB was added. After washing, anavidin-horse radish peroxidase conjugate was added and incubated. Testswere developed with Tris-malcate buffer (TMB)+H₂ O₂ substrate. The testresults are presented in Table 5. The data show the presence of highlevels of IBDV antigen in all bursa in the vaccinate-challenged groupand in the challenged group. No IBDV antigen was detected in thecontrols. IBDV specific antigen could be detected at dilutions of over1/1000, and there does not appear to be differences between vaccinatedand non-vaccinated challenged groups. HVT titers as determined by ELISAwere first detectable at day 7 in four out of the six birds vaccinated.By day 14, six out of six vaccinated birds showed titers to HVT. All sixbirds continued to show HVT titers throughout the experiment. No IBDV SNtiters were seen prior to the challenge. In contrast, analysis of thesesame serum samples by the WESTERN BLOTTING procedure demonstrated theseroconversion of chickens vaccinated with S-HVT-003 to IBDV prior toadministration of the virus challenge. The level of response, however,remains small unless boosted by challenge. Comparison between thevaccinated/challenged and challenged only groups clearly demonstratesthat the level of reactivity by Western blots is much higher in thevaccinated/challenged group. These results show that S-HVT-003 isseroconverting vaccinated birds to IBDV, and suggest that the level ofIBDV specific expression are not high enough to induce a neutralizingresponse in the birds.

S-HVT-003 shows the merit of the vaccine approach the applicants haveinvented. HVT has been engineered to simultaneously express the foreignantigens (β-galactosidase and IBDV antigens) that are recognized in thehost by an immune response directed to these proteins.

                  TABLE 5                                                         ______________________________________                                        Serology: Herpes/IBDV ELISA titer                                               Bleed Date                                                                        Bird #   11/3 11/10                                                                              11/14                                                                              11/24 12/1 12/8 12/15                                                                              12/22                      ______________________________________                                        Vaccinated and Challenged                                                       221      0/0    7/0  5/0  6/0   5/0  5/0  5/0  3/3                             41 0/0 4/0 4/0 1/0 1/0 1/0 1/0 1/3                                            42 0/0 3/0 2/0 1/0 5/0 5/0 5/0 3/2                                            43 0/0 0/0 5/0 5/0 5/0 5/0 3/0 3/2                                            44 0/0 1/0 5/0 1/0 2/0 1/0 1/0 2/4                                            45 0/0 0/0 1/0 1/0 1/0 1/0 1/0 1/3                                         Control                                                                          28      0/0                         0/0                                       38 0/0  0/0                                                                   73 0/0  0/0                                                                   75 0/0  0/0                                                                Challenged only                                                                  40      0/0                         0/0                                       74 0/0  0/0                                                                   39 0/0  0/0                                                                   72 0/0  0/0                                                                ______________________________________                                         Maximum titer level is 9                                                 

EXAMPLE 3

S-HVT-004

S-HVT-004 is a recombinant herpesvirus of turkeys that contains theMarek's disease virus (MDV) glycoprotein A (gA) gene inserted into thelong unique region, and the β-galactosidase (lacZ) gene also inserted inthe long unique region. The MDV antigen is more likely to elicit theproper antigentic response than the HVT equivalent antigen.

The MDV gA (SEQ ID NOS: 8 and 9) gene was cloned by standard DNA cloninggA procedures. An EcoRI restriction fragment had been reported tocontain the MDV gA gene (Isfort et al., 1984) and this fragment wasidentified by size in the DNA clones. The region of the DNA reported tocontain the gA gene was sequenced by applicants and found to contain aglycoprotein gene as expected. The DNA from this gene was used to findthe corresponding gene in HVT by the SOUTHERN BLOTTING OF DNA procedure,and a gene in HVT was identified that contained a very similar sequence.This gene is the same gene previously called gA (Isfort et al., 1984).

For insertion into the genome of HVT, the MDV gA gene was used intactbecause it would have good herpesvirus signal sequences already. ThelacZ gene was inserted into the XhoI fragment in BamHI fragment #16, andthe MDV gA gene was inserted behind lacZ as shown in FIGS. 6A and 6B.Flanking regions in BamHI #16 were used for the homologousrecombination. HVT DNA and plasmid DNA were co-transfected according tothe DNA TRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUS procedureinto primary chick embryo fibroblast (CEF) cells. The virus from thetransfection stock was purified by successive plaque purifications usingthe BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUS procedure. At the end ofthis procedure, when 100% of the plaques were blue, the DNA was analyzedfor the presence of the MDV gA gene. S-HVT-004 is a recombinant virusthat contains both the β-galactosidase gene and the MDV gA geneincorporated into the genome.

FIG. 6C shows the structure of S-HVT-004.

EXAMPLE 4

Newcastle Disease Virus

Newcastle disease virus (NDV) is closely related to PI-3 in overallstructure. Hemagglutinin (HN) and fusion (F) genes of PI-3 wasengineered for expression in IBR (ref). Similarly hemagglutinin (HN) andfusion (F) genes was cloned from NDV for use in the herpesvirus deliverysystem (Herpesvirus of turkeys, HVT).

The procedures that was utilized for construction of herpesvirus controlsequences for expression have been applied to NDV.

Infectious Bronchitis Virus

Infectious bronchitis virus (IBV) is a virus of chickens closely relatedin overall structure to TGE. Major neutralizing antigen of TGE wasengineered for expression in PRV (ref). Similarly major neutralizingantigens was cloned from three strains of IBV: Massachusetts (SEQ IDNOs: 14 and 15), Connecticut (SEQ ID NOs: 18 and 19), and Arkansas-99(SEQ ID NOs: 16 and 17) for use in a herpesvirus delivery system (HVT).

The procedures that was utilized for the construction of herpesviruscontrol sequences for expression have been applied to IBV.

EXAMPLE 5

S-HVT-045

S-HVT-045 is a recombinant herpesvirus of turkeys that contains theMarek's disease virus (MDV) glycoprotein B (gB) gene inserted into theshort unique region. The MDV antigen is more likely to elicit the properantigenic response than the HVT equivalent antigen. S-HVT-045 has beendeposited on Oct. 15, 1992 pursuant to the Budapest Treaty on theInternational Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR 2383.

The MDV gB gene was cloned by standard DNA cloning procedures. The MDVgB gene was localized to a 3.9 kb EcoRI-SalI fragment using anoligonucleotide probe based on the HSV gB sequence in a region found tobe conserved among known herpesvirus gB genes. The restriction map 3.9kb EcoRI-SalI fragment is similar to the published map (Ross et al.,1989).

For insertion into the HVT genome, the MDV gB was used intact because itwould have good herpesvirus signal sequences already. The MDV gB genewas inserted into a cloned 17.15 kb BamHI-EcoRI fragment derived fromthe HVT BamHI #1 fragment. The site used for insertion was the StuI sitewithin HVT US2, previously utilized for the construction of S-HVT-012.The site was initially altered by insertion of a unique HindIII linker,and the MDV gB gene was inserted by standard DNA cloning procedures.Flanking regions in the 17.15 kb BamHI-EcoRI fragment were used,together with the remaining cloned HVT fragments using the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUSES FROM OVERLAPPING SUBGENOMICFRAGMENTS. The virus obtained from the transfection stock was plaquepurified and the DNA was analyzed for the presence of the MDV gB gene.S-HVT-045 is a recombinant virus that contains the MDV gB geneincorporated into the genome at the StuI site in HVT US2 gene.

Testing of Recombinant S-HVT-045

Two studies were conducted to demonstrate the effectiveness of theserecombinant HVT/MDV viruses in protecting against challenge withvirulent Marek's disease virus. In Study A, one-day-old specificpathogen free (SPF) chicks were vaccinated with either S-HVT-045 orS-HVT-046. Seven days post-vaccination, vaccinated chicks, andnon-vaccinated, control chicks were challenged with the highly virulentMD-5 strain of Marek's disease virus. Following a 6-week post-challengeobservation period for clinical signs typical of Marek's disease, allchicks were necropsied and examined for lesions diagnostic of Marek'sdisease. The results, in Table 6, show that both recombinant virusesgave complete protection against a challenge that caused Marek's diseasein 90% of non-vaccinated control chicks.

In a second study, one-day-old chicks were vaccinated either withS-HVT-045 or S-HVT-047. A third group of chicks were vaccinated with aUSDA-licensed, conventional vaccine comprised of HVT and SB-1 viruses.Five days post-vaccination, the vaccinated chicks and a group ofnon-vaccinated, control chicks were challenged with virulent Marek'svirus, strain RB1B. The chicks were observed for 8 weeks for clinicalsigns of Marek's disease, then necropsied and observed for Marek'slesions. This study demonstrated the ability of HVT-045 and HVT-047 toprovide 100% protection against challenge (Table 1). The commercialvaccine gave 96% protection, and 79% of the non-vaccinated chicksdeveloped Marek's disease.

                  TABLE 6                                                         ______________________________________                                        EFFICACY OF RECOMBINANT HVT/MDV VIRUSES TO                                      PROTECT SUSCEPTIBLE CHICKS AGAINST VIRULENT                                   MAREK'S DISEASE VIRUS                                                                    Marek's Protection                                               Vaccine Group                                                                              MD-5 Challenge                                                                            RB1B Challenge                                       ______________________________________                                        S-HVT-045    20/20       24/24                                                  S-HVT-046 20/20 Not Tested                                                    S-HVT-047 Not Tested 24/24                                                    HVT.sup.a Not Tested 24/25                                                    Controls  2/20  5/24                                                        ______________________________________                                         .sup.a Commercial                                                        

EXAMPLE 6

S-HVT-012

S-HVT-012 is a recombinant herpesvirus of turkeys that contains the E.coli β-galactosidase (lacZ) gene inserted into the short unique region.The lacZ gene was used to determine the viability of this insertion sitein HVT [ATCC F-126 ("Calnek")]. S-HVT-012 has been deposited on Oct. 15,1992 pursuant to the Budapest Treaty on the International Deposit ofMicroorganisms for the Purposes of Patent Procedure on with the PatentCulture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR2382.

For insertion into the genome of HVT, the β-galactosidase gene wasintroduced into the unique StuI site of the cloned EcoRI fragment #7 ofHVT, i.e., the fragment containing the StuI site within the US2 gene ofHVT (as described in Methods and Materials). Flanking regions of EcoRIfragment #7 were used for homologous recombination. HVT DNA and plasmidDNA were co-transfected according to the DNA TRANSFECTION FOR GENERATINGRECOMBINANT VIRUS procedure into primary chick embryo fibroblast (CEF)cells. A blue virus obtained from the transfection stock was purified bysuccessive plaque purifications using the BLUOGAL SCREEN FOR RECOMBINANTHERPESVIRUS procedure. At the end of this procedure, when 100% of theplaques were blue, the DNA was analyzed for the presence of the lacZgene. S-HVT-012 is a recombinant virus that contains the lacZ geneincorporated into the genome at the StuI site within the US2 gene ofHVT.

S-HVT-012 may be formulated as a vaccine in the same manner asS-HVT-045. When administered to chickens, such a vaccine providesprotection against Marek's disease virus.

EXAMPLE 7

Sites for Insertion of Foreign DNA into HVT

In order to define appropriate insertion sites, a library of HVT BamHIand EcoRI restriction fragments was generated. Several of theserestriction fragments (BamHI fragments #16 and #13, and EcoRI fragments#6, #7, and #9 (see FIG. 1)) were subjected to restriction mappinganalysis. One unique restriction site was identified in each fragment asa potential insertion site. These sites included XhoI in BamHI fragments#13 and #16, and EcoRI fragment #9 and SalI in EcoRI fragment #6 andStuI in EcoRI fragment #7. A β-galactosidase (lacZ) marker gene wasinserted in each of the potential sites. A plasmid containing such aforeign DNA insert may be used according to the DNA COTRANSFECTION FORGENERATING RECOMBINANT HERPESVIRUSES to CONSTRUCT a HVT containing theforeign DNA. For this procedure to be successful it is important thatthe insertion site be in a region non-essential to the replication ofthe HVT and that the site be flanked with HVT DNA appropriate formediating homologous recombination between virus and plasmid DNAs. Theplasmids containing the lacZ marker gene were utilized in the DNACOTRANSFECTION FOR GENERATING RECOMBINANT HERPESVIRUSES. The generationof recombinant virus was determined by the BLUOGAL SCREEN FORRECOMBINANT HERPESVIRUS. Three of the five sites were successfully usedto generate a recombinant virus. In each case the resulting virus waseasily purified to 100%, clearly defining an appropriate site for theinsertion of foreign DNA. The three homology vectors used to definethese sites are described below.

EXAMPLE 7A

Homology Vector 172-29.31

The homology vector 172-29.31 contains the HVT BamHI #16 fragment and isuseful for the insertion of foreign DNA into HVT. Plasmid 172-29.31contains a unique XhoI restriction site into which foreign DNA may becloned. XhoI site in homology vector 172-29.31 may be used to insertforeign DNA into HVT by the construction of at least three recombinantHVT (see examples 1-3).

The homology vector 172-29.31 was further characterized by DNA sequenceanalysis. The complete sequences of the BamHI #16 fragment wasdetermined. Approximately 2092 base pairs of the adjacent BamHI #13fragment was also determined (see SEQ ID NO: 3). This sequence indicatesthat the open reading frame coding for HVT glycoprotein A (gA) spans theBamHI #16-BamHI #13 junction. The HVT gA gene is homologous to the HSV-1glycoprotein C (gC). The XhoI site interrupts an ORF which lies directlyupstream of the HVT gA gene. This ORF shows amino acid sequence homologyto the PRV p43 and the VZV gene 15. The PRV and VZV genes are thehomologues of HSV-1 UL43. Therefore this ORF was designated as HVT UL43(SEQ ID NO: 5). It should be noted that the HVT UL43 does not exhibitdirect homology to HSV-1 UL43. Although HVT UL43 is located upstream ofthe HVT gC homologue it is encoded on the same DNA strand as HVT gA,where as the HSV-1 UL43 is on the opposite strand relative to HSV-1 gC.The XhoI site interrupts UL43 at approximately amino acid 6, suggestingthat the UL43 gene is non-essential for HVT replication.

EXAMPLE 7B

Homology Vector 435-47.R17

The homology vector 435-47.Rl7 contains the HVT EcoRI #7 fragment and isuseful for the insertion of foreign DNA into HVT. Plasmid 435-47.R17contains a unique HindIII restriction site into which foreign DNA may becloned. The HindIII restriction site in plasmid results from theinsertion of a HindIII linker into the naturally occurring StuI site ofEcoRI fragment #7. HindIII site in homology vector 435-47.R17 may beused to insert foreign DNA into HVT by the construction of at least 25recombinant HVT.

DNA sequence analysis at the StuI indicated that this fragment containsopen reading frames coding for US10, US2, and US3. The StuI siteinterrupts US2 at approximately amino acid 124, suggesting that the US2gene is non-essential for HVT replication.

EXAMPLE 7C

Homology Vector 172-63.1

The homology vector 172-63.1 contains the HVT EcoRI #9 fragment and isuseful for the insertion of foreign DNA into HVT. Plasmid 172-63.1contains a unique XhoI restriction site into which foreign DNA may becloned. XhoI site in homology vector 172-63.1 may be used to insertforeign DNA into HVT by the construction of S-HVT-014 (see example 8).

EXAMPLE 8

S-HVT-014

S-HVT-014 is a recombinant herpesvirus of turkeys that contains the E.coli β-galactosidase (lacZ) gene inserted into the long unique region.The lacZ gene was used to determine the viability of this insertion sitein HVT [ATCC F-126 ("Calnek")].

For insertion into the genome of HVT, the β-galactosidase gene wasintroduced into the unique XhoI site of the cloned EcoRI fragment #9 (asdescribed in Methods and Materials). The XhoI site within the EcoRI #9fragment of the HVT genome is the same site as the XhoI site within theBamHI #10 fragment used for construction recombinant herpesvirues ofturkeys described in Examples 16 through 19. Flanking regions of EcoRIfragment #9 were used for homologous recombination. HVT DNA and plasmidDNA were co-transfected according to the DNA TRANSFECTION FOR GENERATINGRECOMBINANT VIRUS procedure into primary chick embryo fibroblast (CEF)cells. A blue virus obtained from the transfection stock was purified bysuccessive plaque purifications using the BLUOGAL SCREEN FOR RECOMBINANTHERPESVIRUS procedure. At the end of this procedure when 100% of theplaques were blue. S-HVT-014 is a recombinant virus that contains thelacZ gene incorporated into the genome at the XhoI site within the EcoRI#9 fragment of HVT.

S-HVT-014 may be formulated as a vaccine in the same manner asS-HVT-045. When administered to chickens, such a vaccine providesprotection against Marek's disease virus.

EXAMPLE 9

S-HVT-005

S-HVT-005 is a recombinant herpesvirus of turkeys that contains the E.coli β-galactosidase (lacZ) gene inserted into the long unique region.The lacZ gene was used to determine the viability of this insertion sitein HVT [ATCC F-126 ("Calnek")].

For insertion into the genome of HVT, the β-galactosidase gene wasintroduced into an approximately 1300 base pair deletion of the XhoI #9fragment of HVT. The deletion which lies between the unique MluI andEcoRV sites removes the complete coding region of the HVT gA gene (seeSEQ ID NO: 3). Flanking regions of XhoI fragment #9 were used forhomologous recombination. HVT DNA and plasmid DNA were co-transfectedaccording to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUSprocedure into primary chick embryo fibroblast (CEF) cells. A blue virusobtained from the transfection stock was purified by successive plaquepurifications using the BLUOGAL SCREEN FOR RECOMBINANT HERPESVIRUSprocedure. At the end of this procedure, when 100% of the plaques wereblue, the DNA was analyzed for the presence of the lacZ gene. S-HVT-005is a recombinant virus that contains the lacZ gene incorporated into thegenome in place of the deleted gA gene of HVT.

S-HVT-005 may be formulated as a vaccine in the same manner asS-HVT-045. When administered to chickens, such a vaccine providesprotection against Marek's disease virus.

EXAMPLE 10

Marek's Disease Vaccines

Recombinant HVT expressing glycoproteins from Marek's Disease Virus makesuperior vaccines for Marek's Disease. We have constructed severalrecombinant HVT expressing MDV glycoproteins: S-HVT-004 (Example 3),S-HVT-045 (Example 5), S-HVT-046 (Example 10A), S-HVT-047 (Example 10B),S-HVT-062 (Example 10C).

EXAMPLE 10A

S-HVT-046

S-HVT-046 is a recombinant herpesvirus of turkeys that contains theMarek's disease virus (MDV) glycoprotein B (gB) and glycoprotein A (gA)genes inserted into the short unique region. The MDV genes are insertedin the same transcriptional orientation as the US2 gene. The MDVantigens are more likely to elicit the proper antigenic response thanthe HVT equivalent antigen.

S-HVT-046 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and456-17.22 uncut. Insertion of the appropriate DNA was confirmed bysouthern blot analysis.

EXAMPLE 10B

S-HVT-047

S-HVT-047 is a recombinant herpesvirus of turkeys that contains the MDVgB and gA genes inserted into the short unique region. The MDV genes areinserted in the opposite transcriptional orientation as the US2 gene.The MDV antigens are more likely to elicit the proper antigenic responsethan the HVT equivalent antigen.

S-HVT-047 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and456-17.18 uncut. Insertion of the appropriate DNA was confirmed bysouthern blot analysis.

EXAMPLE 10C

S-HVT-062

S-HVT-062 is a recombinant herpesvirus of turkeys that contains the MDVgB, glycoprotein D (gD) and gA genes inserted into the short uniqueregion. The MDV genes are inserted in the same transcriptionalorientation as the US2 gene. The MDV antigens are more likely to elicitthe proper antigenic response than the HVT equivalent antigen. S-HVT-062has been deposited on Feb. 23, 1993 pursuant to the Budapest Treaty onthe International Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR 2401.

S-HVT-062 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 556-60.6 with BamHI and HindIII, and456-17.22 uncut. Insertion of the appropriate DNA was confirmed bysouthern blot analysis.

Testing of Recombinant HVT Expressing MDV Antigens

Two studies were conducted to demonstrate the effectiveness of theserecombinant HVT/MDV viruses in protecting against challenge withvirulent Marek's disease virus. In Study 1, one-day-old specificpathogen free (SPF) chicks were vaccinated with either S-HVT-045,S-HVT-046, or S-HVT-047. Five days post-vaccination, vaccinated chicks,and non-vaccinated, control chicks were challenged with MDV. Following a6-week post-challenge observation period for clinical signs typical ofMarek's disease, all chicks were necropsied and examined for lesionsdiagnostic of Marek's disease. The results, in Table 7, show theserecombinant viruses gave complete protection against a challenge thatcaused Marek's disease in 84% of non-vaccinated control chicks.

In the second study, one-day-old chicks were vaccinated with S-HVT-062.Two more groups of chicks were vaccinated with a USDA-licensed,conventional vaccines comprised of HVT and a combination HVT and SB-1viruses. Five days post-vaccination, the vaccinated chicks and a groupof non-vaccinated, control chicks were challenged with MDV. The chickswere observed for 8 weeks for clinical signs of Marek's disease, thennecropsied and observed for Marek's lesions. This study demonstrated theability of S-HVT-062 to provide 100% protection against challenge (Table7). The commercial vaccines gave 81% and 95% protection, respectivelyand 100% of the non-vaccinated chicks developed Marek's disease.

                  TABLE 7                                                         ______________________________________                                        EFFICACY OF RECOMBINANT HVT/MDV VIRUSES AGAINST                                 VIRULENT MAREK'S VIRUS CHALLENGE                                              Study     Vaccine Group                                                                              Dose.sup.a                                                                           Protection.sup.b                              ______________________________________                                        1       S-HVT-045    2.2 × 10.sup.3                                                                   24/24 (100%)                                      1 S-HVT-046 2.2 × 10.sup.3 20/20 (100%)                                 1 S-HVT-047 2.2 × 10.sup.3 24/24 (100%)                                 1 Controls   7/44 (16%)                                                       1 HVT/SB-1  24/25 (96%)                                                       2 S-HVT-062 7.5 × 10.sup.2 32/32 (100%)                                 2 S-HVT-062 1.5 × 10.sup.3 22/22 (100%)                                 2 Controls  0/20 (0%)                                                         2 HVT.sup.c 7.5 × 10.sup.2 17/21 (81%)                                  2 HVT/SB-1.sup.c 7.5 × 10.sup.2 21/22 (95%)                           ______________________________________                                         .sup.a PFU/0.2 ml.                                                            .sup.b No. protected/Total; Challenge 5 days postvaccination.                 .sup.c Commercial vaccine.                                               

EXAMPLE 11

Bivalent Vaccines Against Newcastle Disease and Marek's Disease

Recombinant HVT expressing proteins from NDV make bivalent vaccinesprotecting against both Marek's Disease and Newcastle disease. Severalrecombinant HVT expressing NDV proteins were constructed S-HVT-007(Example 11A), S-HVT-048 (Example 11B), S-HVT-049 (Example 11C),S-HVT-050 (Example 11D),and S-HVT-106 (Example 11E).

EXAMPLE 11A

S-HVT-007

S-HVT-007 is a recombinant herpesvirus of turkeys that contains a E.coli lacZ NDV HN hybrid protein gene under the control of the PRV gXpromoter and the NDV F gene under the control of the HSV-1 α4 promoterinserted into the long unique region. The NDV genes are inserted in thesame transcriptional orientation as the UL43 gene.

To construct S-HVT-007, HVT DNA and the plasmid 255-18.B16 wereco-transfected according to the DNA TRANSFECTION FOR GENERATINGRECOMBINANT VIRUS procedure into primary chick embryo fibroblast (CEF)cells. A blue virus obtained from the transfection stock was purified bysuccessive plaque purifications using the BLUOGAL SCREEN FOR RECOMBINANTHERPESVIRUS procedure. At the end of this procedure, when 100% of theplaques were blue.

EXAMPLE 11B

S-HVT-048

S-HVT-048 is a recombinant herpesvirus of turkeys that contains the MDVgB and gA genes and the NDV F gene under the control of the HCMVimmediate early promoter inserted into the short unique region. The MDVand NDV genes are inserted in the same transcriptional orientation asthe US2 gene.

S-HVT-048 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones-and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and535-70.3 uncut. Insertion of the appropriate DNA was confirmed bysouthern blot analysis.

EXAMPLE 11C

S-HVT-049

S-HVT-049 is a recombinant herpesvirus of turkeys that contains the MDVgB and gA genes and the NDV HN gene under the control of the PRV gXpromoter inserted into the short unique region. The MDV and NDV genesare inserted in the same transcriptional orientation as the US2 gene.

S-HVT-049 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and549-62.10 uncut. Insertion of the appropriate DNA was confirmed bysouthern blot analysis.

EXAMPLE 11D

S-HVT-050

S-HVT-050 is a recombinant herpesvirus of turkeys that contains the MDVgB and gA genes and the NDV HN (SEQ ID NOs: 10 and 11) and F (SEQ IDNOs: 12 and 13) genes. The NDV genes are under the control of the PRV gXand HCMV immediately promoters respectively. All four genes are insertedinto the short unique region in the same transcriptional orientation asthe US2 gene.

S-HVT-050 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and549-24.15 uncut. Insertion of the appropriate DNA was confirmed bysouthern blot analysis. S-HVT-050 has been deposited on Feb. 23, 1993pursuant to the Budapest Treaty on the International Deposit ofMicroorganisms for the Purposes of Patent Procedure with the PatentCulture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR2400.

EXAMPLE 11E

S-HVT-106

S-HVT-106 is a recombinant herpesvirus of turkeys that contains the MDVgA, gB, gD genes and the NDV HN and F genes. The NDV genes are under thecontrol of the PRV gX and HCMV immediately promoters respectively. Allfive genes are inserted into the short unique region in the sametranscriptional orientation as the US2 gene.

S-HVT-106 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and633-13.27 uncut.

Testing of Recombinant HVT Expressing NDV Antigens

Two studies were conducted to demonstrate the effectiveness of theserecombinant HVT/MDV/NDV viruses in protecting against challenge withvirulent Newcastle and Marek's disease viruses. In Study 1, one-day-oldspecific pathogen free (SPF) chicks were vaccinated with eitherS-HVT-048, S-HVT-049, S-HVT-050, or a USDA-licensed, conventionalvaccine comprised of NDV B1/B1 virus. Three weeks post-vaccination,vaccinated chicks, and non-vaccinated, control chicks were challengedwith NDV. Birds were then observed for clinical signs of disease. Theresults, in Table 8, show these recombinant viruses (S-HVT-048 andS-HVT-050) gave complete protection against a challenge that causedNewcastle disease in 100% of non-vaccinated control chicks. Recombinantvirus S-HVT-049 gave partial protection against Newcastle disease.

In the second study, one-day-old chicks were vaccinated with S-HVT-050.Two more groups of chicks were vaccinated with a USDA-licensed,conventional vaccines comprised of HVT and a combination HVT and SB-1viruses. Five days post-vaccination, the vaccinated chicks and a groupof non-vaccinated, control chicks were challenged with MDV. The chickswere observed for 8 weeks for clinical signs of Marek's disease, thennecropsied and observed for Marek's lesions. This study demonstrated theability of S-HVT-050 to provide protection greater than the commercialMarek's disease vaccines.

                  TABLE 8                                                         ______________________________________                                        EFFICACY OF RECOMBINANT HVT/MDV/NDV VIRUSES                                     AGAINST VIRULENT NEWCASTLE AND MAREK'S DISEASE                                VIRUS CHALLENGE                                                                   Vaccine     Protection (%)                                              Study Group       Dose.sup.a                                                                              NDV.sup.b                                                                              MDV.sup.c                                ______________________________________                                        1     S-HVT-048   4.0 × 10.sup.4                                                                    19/19 (100)                                         1 S-HVT-049 3.0 × 10.sup.4 4/20 (20)                                    1 S-HVT-050 1.5 × 10.sup.4 20/20 (100)                                  1 Controls  0/20 (0)                                                          1 NDV B1/B1.sup.d  18/18 (100)                                                2 S-HVT-050 7.5 × 10.sup.2  13/14 (93)                                  2 S-HVT-050 1.5 × 10.sup.3  16/17 (94)                                  2 Controls    5/23 (22)                                                       2 HVT.sup.d   20/26 (77)                                                      2 HVT/SB-1.sup.d   10/12 (83)                                               ______________________________________                                         .sup.a PFU/0.2 ml.                                                            .sup.b No. protected/Total; Challenge 3 weeks postvaccination.                .sup.c No. protected/Total; Challenge 5 days postvaccination.                 .sup.d Commercial vaccine.                                               

EXAMPLE 12

Bivalent Vaccines Against Infectious Laryngotracheitis and Marek'sDisease

Recombinant HVT expressing glycoproteins from ILT virus make bivalentvaccines protecting against both Marek's disease and infectiouslaryngotracheitis. Several recombinant HVT expressing ILT virusglycoproteins S-HVT-051 (Example 12A), S-HVT-052 (Example 12B), andS-HVT-104 (Example 11C) were constructed.

EXAMPLE 12A

S-HVT-051

S-HVT-051 is a recombinant herpesvirus of turkeys that contains the ILTvirus gB gene inserted into the short unique region. The ILT gene isinserted in the same transcriptional orientation as the US2 gene.

S-HVT-051 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and528-11.34 uncut. Insertion of the appropriate DNA was confirmed bysouthern blot analysis.

EXAMPLE 12B

S-HVT-052

S-HVT-052 is a recombinant herpesvirus of turkeys that contains the ILTvirus gD gene inserted into the short unique region. The ILT gene isinserted in the opposite transcriptional orientation as the US2 gene.S-HVT-052 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 437-26.26 with BamHI and HindIII, and528-03.37 uncut. Insertion of the appropriate DNA was confirmed bysouthern blot analysis.

EXAMPLE 12C

S-HVT-104

S-HVT-104 is a recombinant herpesvirus of turkeys that contains sixforeign genes. The MDV gA, gB, and gD genes are inserted in the uniqueshort region in the same transcriptional orientation as the US2 gene. AnE. coli lacZ marker gene and the ILT gB and gD genes are inserted inBamHI #16 region in the same transcriptional orientation as the UL43gene.

To construct S-HVT-104, DNA from S-HVT-062 and the plasmid 634-29.16were co-transfected according to the DNA TRANSFECTION FOR GENERATINGRECOMBINANT VIRUS procedure into primary chick embryo fibroblast (CEF)cells.

Testing of Recombinant HVT Expressing ILT Antigens

The following study was conducted to demonstrate the effectiveness ofthese recombinant HVT/ILT viruses in protecting against challenge withvirulent Infectious Laryngotracheitis virus. One-day-old specificpathogen free (SPF) chicks were vaccinated with either S-HVT-051,S-HVT-052, a combination of S-HVT-051 and S-HVT-052, or a USDA-licensed,conventional vaccine comprised of ILT virus. Two to three weekspost-vaccination, vaccinated chicks, and non-vaccinated, control chickswere challenged with ILT. Birds were then observed for clinical signs ofdisease. The results, in Table 9, show these recombinant viruses(S-HVT-051 and S-HVT-052) gave protection against challenge with ILTvirus comparable to a commercial ILT vaccine.

Animals vaccinated with the vaccines described here may be easilydifferentiated from animals infected with virulent ILT. This isaccomplished by testing the suspect birds for antibodies to any ILTantigens other than gB or gD. Examples of such antigens are ILTglycoproteins C, E, and G. Vaccinated, uninfected birds will be negativefor these antigens whereas infected birds will be positive.

                  TABLE 9                                                         ______________________________________                                        EFFICACY OF RECOMBINANT HVT/ILT VIRUSES AGAINST                                 VIRULENT INFECTIOUS LARYNGOTRACHEITIS                                         VIRUS CHALLENGE                                                                  Vaccine Group Dose.sup.a                                                                             Protection.sup.b                                  ______________________________________                                        S-HVT-051      2.1 × 10.sup.3                                                                   28/30 (93%)                                             S-HVT-052 1.7 × 10.sup.3 29/29 (100%)                                   S-HVT-051 + 2.1 × 10.sup.3 24/24 (100%)                                 S-HVT-052 1.7 × 10.sup.3                                                Controls  2/30 (7%)                                                           ILT.sup.c  29/30 (97%)                                                      ______________________________________                                         .sup.a PFU/0.2 ml.                                                            .sup.b No. protected/Total; Challenge 2-3 weeks postvaccination.              .sup.c Commercial vaccine.                                               

EXAMPLE 13

Bivalent Vaccines Against Infectious Bursal Disease and Marek's Disease

Recombinant HVT expressing proteins from IBDV make bivalent vaccinesprotecting against both Marek's Disease and infectious bursal disease.Several recombinant HVT expressing IBDV proteins were constructed. Theseviruses include S-HVT-003 (example 2) and S-HVT-096.

S-HVT-096 is a recombinant herpesvirus of turkeys that contains the IBDVVP2 gene, under the control of the HCMV immediate early promoter,inserted into the short unique region. The IBDV gene is inserted in thesame transcriptional orientation as the US2 gene.

S-HVT-096 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 556-60.6 with BamHI, and 602-57.F1uncut. Insertion of the appropriate DNA was confirmed by southern blotanalysis.

S-HVT-096 was assayed for expression of VP2 by black plaque and westernblot analysis. Both assays indicated that the virus was expressing highlevels of protein which reacts specifically with an IBDV neutralizingmonoclonal antibody. This virus will be useful as a vaccine againstinfectious bursal disease.

EXAMPLE 14

Bivalent Vaccines Against Infectious Bronchitis and Marek's Disease

S-HVT-066 is a recombinant herpesvirus of turkeys that contains the MDVgB, gD and gA genes and the IBV spike and matrix genes. The IBV spikeand matrix genes are under the control of the HCMV immediate early andPRV gX promoters respectively. All five genes are inserted into theshort unique region. The MDV and IBV genes are inserted in the sametranscriptional orientation as the US2 gene.

S-HVT-066 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS. The followingcombination of subgenomic clones and enzymes were used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 407-32.1C1 with NotI,437-26.24 with BamHI and HindIII, 556-60.6 with BamHI, and 567-72.1Duncut. Insertion of the appropriate DNA was confirmed by southern blotanalysis.

S-HVT-066 was assayed for expression of the IBV spike protein by blackplaque and western blot analysis. Both assays indicated that the viruswas expressing high levels of protein which reacts specifically with anIBV neutralizing monoclonal antibody. This virus will be useful as avaccine against infectious bronchitis.

EXAMPLE 15

Vaccines Utilizing HVT to Express Antigens from Various Pathogens

Anticipate that antigens from the following pathogens may also beutilized to develop poultry vaccines: Chick anemia virus (agent), Avianencephalomyelitis virus, Avian reovirus, Avian paramyxoviruses, Avianinfluenza virus, Avian adenovirus, Fowl pox virus, Avian coronavirus,Avian rotavirus, Salmonella spp E. coli, Pasteurella spp, Haemophilusspp, Chlamydia spp, Mycoplasma spp, Campylobacter spp, Bordetella spp,Poultry nematodes, cestodes, trematodes, Poultry mites/lice, Poultryprotozoa (Eimeria spp, Histomonas spp, Trichomonas spp).

EXAMPLE 16

Trivalent vaccines against Infectious Laryngotracheitis, Marek's Diseaseand Newcastle's Disease and bivalent vaccines against InfectiousLaryngotracheitis and Marek's Disease are described. Superior protectionagainst Infectious Laryngotracheitis is achieved with a vaccinecombining S-HVT-123 (expressing ILTV gB and gD) with S-HVT-138, 139, or140 (expressing ILTV gD and gI).

EXAMPLE 16A

S-HVT-123

S-HVT-123 is a recombinant herpesvirus of turkeys that contains the ILTvirus gB and gD genes inserted into an XhoI site converted to a NotIsite in the EcoR1 #9 (BamHI #10) fragment of the HVT genome (FIGS. 13Band 15; SEQ ID NO: 48). S-HVT-123 further contains the MDV gA, gD, andgB genes inserted into a unique StuI site converted into a HindIII sitein the HVT US2 gene. The ILTV genes and the MDV genes each use their ownrespective promoters. S-HVT-123 is useful as a vaccine in poultryagainst Infectious Laryngotracheitis and Marek's Disease.

S-HVT-123 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, 721-38.1J uncut, 729-37.1with AscI.

EXAMPLE 16B

S-HVT-138

S-HVT-138 is a recombinant herpesvirus of turkeys that contains the ILTvirus gD and gI genes inserted into a unique XhoI site converted to aPacI site in the EcoR1 #9 (BamHI #10) fragment of the HVT genome (FIGS.13A and 15). The ILTV gD and gI genes are in the oppositetranscriptional orientation to the open reading frame (ORF A) within theEcor1 #9 (BamHI #10) fragment of the HVT genome (FIG. 14; SEQ ID NOs:48, 50). The ILTV gD and gI genes are expressed as overlappingtranscripts from endogenous ILTV promoters, and share their ownendogenous polyadenylation signal.

S-HVT-138 is useful as a vaccine in poultry against InfectiousLaryngotracheitis and Marek's Disease.

S-HVT-138 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, 711-92.1A uncut, 415-09.Bμlwith BamHI. Sera from S-HVT-138 vaccinated chickens reacts on Westernblots with ILTV gI protein indicating that the S-HVT-138 vaccineexpressed the ILTV protein and does elicit an immune response in birds.S-HVT-138 vaccinated chickens were protected from challenge by virulentinfectious laryngotracheitis virus.

EXAMPLE 16C

S-HVT-139

S-HVT-139 is a recombinant herpesvirus of turkeys that contains the ILTvirus gD and gI genes inserted into a unique XhoI site converted to aPacI site in the EcoR1 #9 (BamHI #10) fragment of the HVT genome. TheILTV gD and gI genes are in the opposite transcriptional orientation tothe open reading frame (ORF A) within the EcoR1 #9 (BamHI #10) fragmentof the HVT genome (FIG. 13A and 15; SEQ ID NO: 48, 50). S-HVT-139further contains the MDV gA, gD, and gB genes are inserted into theunique StuI site converted into a HindIII site in the HVT US2 gene. TheILTV gD and gI genes are expressed as overlapping transcripts from theirwon respective endogenous ILTV promoters, and the MDV genes are alsoexpressed from their own endogenous promoters. S-HVT-139 is useful as avaccine in poultry against Infectious Laryngotracheitis and Marek'sDisease.

S-HVT-139 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, 711-92.1A uncut, 721-38.1Juncut.

EXAMPLE 16D

S-HVT-140

S-HVT-140 is a recombinant herpesvirus of turkeys that contains the ILTvirus gD and gI genes inserted into a unique XhoI site converted to aPacI site in the EcoR1 #9 (BamHI #10) fragment of the HVT genome (FIGS.13A and 15). The ILTV gD and gI genes are in the oppositetranscriptional orientation to the open reading frame (ORF A) within theEcoR1 #9 (BamHI #10) fragment of the HVT genome (FIG. 14; SEQ ID NO: 48,50). S-HVT-140 further contains the MDV gA, gD, and gB genes and the NDVF and HN genes inserted into a unique StuI site converted into a HindIIIsite in the HVT US2 gene. The ILTV gD and gI genes are expressed asoverlapping transcripts from their own respective endogenous ILTVpromoters, and the MDV genes are also expressed from their ownrespective endogenous MDV promoters. The NDV F gene is transcribed fromthe HCMV immediate early promoter, and the NDV HN gene is transcribedfrom the PRV gX promoter. S-HVT-140 is useful as a vaccine in poultryagainst Infectious Laryngotracheitis, Marek's Disease, and Newcastle'sDisease.

S-HVT-140 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, 711-92.1A uncut, 722-60.E2uncut.

EXAMPLE 17

Trivalent vaccines against Infectious Bursal Disease, Marek's Diseaseand Newcastle's Disease and bivalent vaccines against Infectious BursalDisease and Marek's Disease are described.

EXAMPLE 17A

HVT-126

S-HVT-126 is a recombinant herpesvirus of turkeys that contains the IBDVVP2 gene inserted into an XhoI site converted to a PacI site in theEcoR1 #9 (BamHI #10) fragment in the HVT genome (FIGS. 13A and 15). TheIBDV gene is in the same transcriptional orientation as the open readingframe (ORF A) within the EcoR1 #9 (BamHI #10) fragment of the HVT genome(FIG. 14; SEQ ID NO: 48, 50). The IBDV VP2 gene is expressed from anIBRV VP8 promoter. S-HVT-126 is useful as a vaccine in poultry againstInfectious Bursal Disease and Marek's Disease.

S-HVT-126 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, 706-57.A3 uncut, 415-09.BA1with BamHI.

EXAMPLE 17B

HVT-137

S-HVT-137 is a recombinant herpesvirus of turkeys that contains the IBDVVP2 gene inserted into a uniqe XhoI site converted to a PacI site in theEcoR1 #9 (BamHI #10) fragment in the HVT genome (FIGS. 13A and 15). TheIBDV gene is in the same transcriptional orientation as the open readingframe (ORF A) within the EcoR1 #9 (BamHI #10) fragment of the HVT genome(FIG. 14; SEQ ID NO: 48, 50). S-HVT-137 further contains the MDV gA, gD,and gB genes inserted into a unique StuI site converted into a HindIIIsite in the HVT US2 gene. The IBDV VP2 gene is expressed from an IBRVVP8 promoter. The MDV genes are expressed from their own respectiveendogenous MDV promoters. S-HVT-137 is useful as a vaccine in poultryagainst Infectious Bursal Disease and Marek's Disease.

S-HVT-137 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, 706-57.A3 uncut, 721-38.1Juncut.

EXAMPLE 17C

HVT-143

S-HVT-143 is a recombinant herpesvirus of turkeys that contains the IBDVVP2 gene inserted into a unique XhoI site converted to a PacI site inthe EcoR1 #9 (BamHI #10) fragment of the HVT genome (FIGS. 13A and 15).The IBDV gene is in the same transcriptional orientation as the openreading frame (ORF A) within the EcoR1 #9 (BamHI #10) fragment of theHVT genome (FIG. 14; SEQ ID NO: 48, 50). S-HVT-143 further contains theMDV gA, gD, and gB genes and the NDV F and HN genes inserted into aunique StuI site converted into a HindIII site in the HVT US2 gene. TheIBDV VP2 gene is expressed from an IBRV VP8 promoter. The MDV genes areexpressed from their own respective endogenous MDV promoters. The NDV Fgene is transcribed from the HCMV immediate early promoter, and the NDVHN gene is transcribed from the PRV gX promoter. S-HVT-143 is useful asa vaccine in poultry against Infectious Bursal Disease, Marek's Disease,and Newcastle's Disease.

S-HVT-143 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, 706-57.A3 uncut, 722-60.E2uncut.

EXAMPLE 18

HVT-128

S-HVT-128 is a recombinant herpesvirus of turkeys that contains the NDVHN and F genes inserted into a unique XhoI site converted to a PacI sitein the EcoR1 #9 (BamHI #10) fragment of the HVT genome (FIGS. 13A and15). S-HVT-128 further contains the MDV gA, gD, and gB genes insertedinto a unique StuI site converted into a HindIII site in the HVT US2gene. The NDV HN gene is expressed from the PRV gX promoter and the NDVF gene is expressed from the HCMV immediate early promoter. The MDVgenes are expressed from the endogenous MDV promoters. S-HVT-128 isuseful as a vaccine in poultry against Newcastle's Disease and Marek'sDisease.

S-HVT-128 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, and 717-38.12 uncut. To amixture of these six cosmids was added a limiting dilution of arecombinant HVT virus containing the MDV gA, gD, and gB genes insertedinto the unique short region (see HVT-062) and the PRV gX promoter-lacZgene inserted into an XhoI site converted to a NotI site in the EcoR1 #9(BamHI #10) fragment within the unique long region of HVT. A recombinantvirus S-HVT-128 was selected which was lac Z negative.

EXAMPLE 18B

HVT-136

S-HVT-136 is a recombinant herpesvirus of turkeys that contains the NDVHN and F genes inserted into an XhoI site converted to a PacI site inthe EcoR1 #9 (BamHI #10) fragment within the unique long region of HVT.(FIG. 14; SEQ ID NOs: 48 and 50) The NDV HN gene is expressed from thePRV gX promoter and the NDV F gene is expressed from the HCMV immediateearly promoter. S-HVT-136 is useful as a vaccine in poultry againstNewcastle's disease and Marek's disease.

S-HVT-136 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, and 717-38.12 uncut, and415-09.BA1 with BamHI.

EXAMPLE 19

S-HVY-145

Recombinant HVT/MDV Chimeric Virus

S-HVY-145 is a recombinant virus vaccine containing MDV and HVT genomicsequences which protects against Marek's disease is produced bycombining cosmids of MDV genomic DNA containing genes coding for therelevant protective antigens of virulent MDV serotype 2 and cosmids ofHVT genomic DNA according to the PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The resulting virusis a vaccine that ahs the protective immune respnse to virulent MDVserotype 2 and the attenuated growth characteristics of the HVT. In oneembodiment, a chimeric virus vaccine containing the MDV genes of theunique short and the HVT genes of the unique long is useful as a vaccineagainst Marek's disease in chickens. The MDV protective antigens withinthe unique short (gD, gE, and gI) elicit a protective immune response toMDV, while the virulence elements present in the unique long of MDV(55,56, 57) are replaced by the attenuating uniuqe long sequences ofHVT. The result is an attenuated virus vaccine which protects againstMarek's disease. Multivalent protection against Marek's disease,infectious laryngotracheitis, infectious vursal disease, Newcastle'sdises, or another poultry pathogen is achieved by inserting the ILTVgB,gD, and gI genes, the IBDV VP2 gene, the NDV HN and F genes, or anantigen gene from a poultry pathogen into an XhoI site converted to aPacI site or NotI site in the EcoR1 #9 (BamHI #10) fragment within theuniuqe long region of HVT/MDV recombinant virus (FIGS. 13 and 15).

A cosmid was constructed containing the entir MDV unique short region.MDV genomic DNa contains several SmaI sites in the uniuqe long andinternal and terminal repeats of the virus, but no SmaI sites within theunique short of the virus. The entire unique short region of MDV wasisolated by a partial restriction digestion of MDV genomic DNa withSmaI. A DNA fragment approximately 29,000 to 33,000 base pairs wasisolated and cloned into a blunt ended site of the cosmid vector pWE15.To generate HVY-145, a recombinant HVT/MDV chimeric virus, the cosmidcontaining the MDV unique short region was combined with cosmidscontaining the HVT unique long region according to the PROCEDURE FORGENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMICFRAGMENTS. The following combination of subgenomic clones and enzymeswere used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 withNotI, 407-32.1C1 with NotI, and 739-27.16 with NotI.

The resulting virus vaccine provides superior protection against Marek'sdisease or as a multivalent vaccine against Marek's disease andinfectious laryngotracheitis, infectious bursal disease, Newcastle'sdisease, or another poultry pathogen. This vaccine is superior becauseexpression of MDV genes in the HVT/MDV chimera vaccine is safer andprovides better protection against Marek's disease than vaccinespresently available containing HVT and MDV type 1 (SB-1) or HVT alone.Secondly, one can demonstrate expression of the MDV glycoprotein gens inthe absence of the homologous HVT genes for both diagnostic andregulatory purposes. This is useful since antibodies to an MDVglycoprotein will cross react with the homologous HVT glycoprotein.Finally, a recombinant HVT/MDV virus which contains a single copy ofeach glycoprotein gene is more stable that a recombinant viruscontaining two copies of a homologous glycoprotein gene from HVT and MDVwhich may delete by homologous recombination.

In an alternative embodiment, cosmids containing MDV protective antigengenes from the unique long (MDV gB and gC) are combined with cosmidscontaining HVT gene sequences from the unique short and the unique long,effectively avoiding the MDV virulence genes at the unique long/internalrepeat junction and the unique long/terminal repeat junction (55, 56,and 57).

SB-1 strain is an MDV serotype 1 with attenuated pathogenicity.Vaccination with a combination of HVT and SB-1 live viruses protectsagainst virulent MDV challenge better than vaccination with either virusalone. In an alternative embodiment of the present invention, arecombinant virus vaccine comprises protective antigen genes of thevirulent MDV serotypes 2 combined with the attenuating genes of thenon-virulent MDV serotypes 1 and 3, such as SB-1 and HVT. The genomicDNA corresponding to the unique long region is contributed by the SB-1serotype. The genomic DNA corresponding to the unique short region iscontributed by the HVT serotype. Three major glycoprotein antigens (gB,gA and gD) from the MDV serotype 2 are inserted into the unique shortregion of the virus.

The recombinant virus is constructed utilizing HVT subgenomic clones672-01.A40, 672-07.C40 and 721-38.1J to reconstruct the unique shortregion. Subgenomic clone 721-38.1J contains an insertion of the MDV gB,gA, and gD genes. A large molar excess of these clones is cotransfectedwith a sub-infectious dose of Sb-1 genomic DNA. To determine theappropriate sub-infectious dose, transfection of the SB-1 is titrateddown to a dose which no longer yields virus plaques in cell culture.Such a dose contains sub-genomic fragments spanning the unique longregion of SB-1 which recombine with the HVT unique short subgenomicclones. Therefore, a virus resulting from recombination betweenoverlapping homologous regions of the SB-1 and HVT subgenomic fragmentsis highly favored. Alternatively, SB-1 genomic fragments from the uniquelong region are subcloned into cosmid vectors. A recombinant viruscontaining the Sb-1 unique long the HVT unique short with the MDV, gB,gA, and gD genes were produced using the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thisprocedure is also used with an HVT subgenomic clone to insert antigengenes from other avian pathogens including but not limited to infectiouslaryngotracheitis virus, Newcastle's disease virus and infectious bursaldisease virus.

EXAMPLE 20

Recombinant HVT expressing chicken myelomonocytic growth factor (cMGF)or chicken interferon (cIFN) are useful as vaccines against Marek'sdisease virus and are also useful to enhance the immune response againstother diseases of poultry. Chicken myelomonocytic growth factor (cMGF)is related to mammalian G-CSF and interleukin-6 protein (58), andchicken interferon (cIFN) is homologous to mammalian type 1 interferon(59) interferon. When used in combination with vaccines described inprevious examples, S-HVT-144 or HVT expressing cIFN are useful toprovide enhanced mucosal, humoral, or cell mediated immunity againstavian disease-causing viruses including, but not limited to, Marek'sdisease virus, Newcastle disease virus, infectious laryngotracheitisvirus, infectious bronchitis virus, infectious bursal disease virus.Recombinant HVT expressing cMGF or cIFN are useful provide enhancedimmunity against avian disease causing organisms described in Example15.

EXAMPLE 20A

S-HVT-144

S-HVT-144 is a recombinant herpesvirus of turkeys that contains thechicken myelomonocytic growth factor (cMGF) gene inserted into an XhoIsite converted to a PacI site in the EcoR1 #9 fragment within the uniquelong region of HVT. The cMGF gene is in the opposite transcriptionalorientation to the open reading frame (ORF A) within the EcoR1 #9fragment of the HVT genome (FIG. 14; SEQ ID NOs: 48 and 50). The cMGFgene is expressed from a human cytomegalovirus immediate early promoter.S-HVT-144 is useful as a vaccine in poultry against Marek's Disease.

S-HVT-144 was constructed according to the PROCEDURE FOR GENERATINGRECOMBINANT HERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. Thefollowing combination of subgenomic clones and enzymes were used:407-32.2C3 with NotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI,672-07.C40 with NotI, 672-01.A40 with NotI, 751-87.A8 with Asc I,415-09.BA1 with BamHI.

EXAMPLE 20B

Recombinant HVT Expressing Chicken Interferon

A recombinant herpesvirus of turkeys contains the chicken interferon(CIFN) gene inserted into an XhoI site converted to a PacI site in theEcoR1 #9 fragment within the unique long region of HVT. The cIFN gene isexpressed from a human cytomegalovirus immediate early promoter.Recombinant HVT expressing cIFN is useful as a vaccine in poultryagainst Marek's Disease.

Recombinant HVT expressing cIFN is constructed according to thePROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM OVERLAPPINGSUBGENOMIC FRAGMENTS. The following combination of subgenomic clones andenzymes were used: 407-32.2C3 with NotI, 172-07.BA2 with BamHI,407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 with NotI,761-07. μl with Asc I, 415-09.Bμl with BamHI.

Recombinant HVT expressing avian cytokines is combined with HVTexpressing genes for avian disease antigens to enhance immune response.Additional cytokines that are expressed in HVT and have immunestimulating effects include, but not limited to, transforming growthfactor beta, epidermal growth factor family, fibroblast growth factors,hepatocyte growth factor, insulin-like growth factors, B-nerve growthfactor, platelet-derived growth factor, vascular endothelial growthfactor, interleukin 1, IL-1 receptor antagonist, interleukin 2,interleukin 3, interleukin 4, interleukin 5, interleukin 6, IL-6 solublereceptor, interleukin 7, interleukin 8, interleukin 9, interleukin 10,interleukin 11, interleukin 12, interleukin 13, angiogenin, chemokines,colony stimulating factors, granulocyte-macrophage colony stimulatingfactors, erythropoietin, interferon, interferon gamma, leukemiainhibitory factor, oncostatin M, pleiotrophin, secretory leukocyteprotease inhibitor, stem cell factor, tumor necrosis factors, andsoluble TNF receptors. These cytokines are from avian species or otheranimals including humans, bovine, equine, feline, canine or porcine.

EXAMPLE 20C

Recombinant HVT Expressing Marek's Disease Virus Genes and ChickenInterferon Gene

A recombinant herpesvirus of turkeys contains the chicken interferon(cIFN) gene inserted into an Xhol site converted to a PacI site in theEcoR1 #9 fragment within the unique long region of HVT and furthercontains the MDV gA, gD, and gB genes inserted into a unique StuI siteconverted into a HindIII site in the HVT US2 gene. The cIFN gene isexpressed from an human cytomegalovirus immediate early promoter. TheMDV genes are expressed from the endogenous MDV promoters. RecombinantHVT expressing cIFN and MDV gA, gB, and gD is useful as a vaccine withan enhanced immune response in poultry against Marek's Disease.

Recombinant HVT expressing MDV genes and the cIFN gene is constructedaccording to the PROCEDURE FROM GENERATING RECOMBINANT HERPESVIRUS FROMOVERLAPPING SUBGENOMIC FRAGMENTS. The following combination ofsubgenomic clones and enzymes are used: 407-32.2C3 with NotI, 172-07.BA2with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 withNotI, 761-07.A1 with Asc I, 721-38.1J uncut.

EXAMPLE 20D

Recombinant HVT Expressing Marek's Disease Virus Genes, NewcastleDisease Virus Genes and Chicken Interferon Gene

A recombinant herpesvirus of turkeys contains the chicken interferon(cIFN) gene inserted into an XhoI site converted to a PacI site in theEcoR1 #9 fragment within the unique long region of HVT and furthercontains the MDV gA, gD, and gB genes and NDV HN and F genes insertedinto a unique StuI site converted into a HindIII site in the HVT US2gene. The cIFN gene is expressed from an human cytomegalovirus immediateearly promoter. The MDV genes are expressed from the endogenous MDVpromoters. The NDV HN gene is under the control of the PRV gX promoter,and the NDV F gene is under the control of the HCMV immediate earlypromoter. Recombinant HVT expressing cIFN and MDV gA, gB, and gD isuseful as a vaccine with an enhanced immune response in poultry againstMarek's Disease and Newcastle disease.

Recombinant HVT expression MDV genes, NDV genes and cIFN is constructedaccording to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROMOVERLAPPING SUBGENOMIC FRAGMENTS. The following combination ofsubgenomic clones and enzymes are used: 407-32.2C3 with NotI, 172-07.BA2with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 withNotI, 761-07.Al with Asc I, 722-60.E2 uncut.

EXAMPLE 20E

Recombinant HVT Expressing Marek's Disease Virus Genes and ChickenMyelomonocytic Growth Factor Gene

A recombinant herpesvirus of turkeys contains the chicken myelomonocyticgrowth factor (cMGF) gene inserted into and XhoI site converted to aPacI site in the EcoR1 #9 fragment within the unique long region of HVTand further contains the MDV gA, gD, and gB genes inserted into a uniqueStuI site converted into a HindIII site in the HVT US2 gene. The cMGFgene is expressed from a human cytomegalovirus immediate early promoter.The MDV genes are expressed from the endogenous MDV promoters.Recombinant HVT expression cMGF and MDV gA, gB, and gD is useful as avaccine with an enhanced immune response in poultry against Marek'sDisease.

Recombinant HVT expressing the cMGF gene and MDV genes is constructedaccording to the PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROMOVERLAPPING SUBGENOMIC FRAGMENTS. The following combination ofsubgenomic clones and enzymes are used: 407-32.2C3 with NotI, 172-07.BA2with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI, 672-01.A40 withNotI, 751-87.A8 with Asc I, 721-38.1J uncut.

EXAMPLE 20F

Recombinant HVT Expressing Marek's Disease Virus Genes, NewcastleDisease Virus Genes and Chicken Myelomonocytic Growth Factor Gene

A recombinant herpesvirus of turkeys contains the chicken myelomonocyticgrowth factor (cGMF) gene inserted into an XhoI site converted to a PacIsite in the EcoR1 #9 fragment within the unique long region of HVT andfurther contains the MDV gA, gD, and gB genes and NDV HN and F genesinserted into a unique StuI site converted into a HindIII site in theHVT US2 gene. The cGMF gene is expressed from an human cytomegalovirusimmediate early promoter. The MDV genes are expressed from theendogenous MDV promoters. The NDV HN gene is under the control of thePRV gX promoter, and the NDV F gene is under the control of the HCMVimmediate early promoter. Recombinant HVT expressing cIFN and MDV gA, gBand gD is useful as a vaccine with an enhanced immune response inpoultry against Marek's Disease and Newcastle disease.

Recombinant HVT expressing MDV genes, NDV genes and the cGMF gene isconstructed according to the PROCEDURE FOR GENERATING RECOMBINANTHERPESVIRUS FROM OVERLAPPING SUBGENOMIC FRAGMENTS. The followingcombination of subgenomic clones and enzymes are used: 407-32.2C3 withNotI, 172-07.BA2 with BamHI, 407-32.5G6 with NotI, 672-07.C40 with NotI,672-01.A40 with NotI, 751-87.A8 uncut, 722-60.E2 uncut.

EXAMPLE 21

Recombinant Herpesvirus of Turkeys Expressing Antigens from DiseaseCausing Microorganisms

Recombinant herpesvirus of turkeys (HVT) is useful for expressingantigens from disease causing microorganisms from animals in addition toavian species. Recombinant HVT is useful as a vaccine in animalsincluding but not limited to humans, equine, bovine, porcine, canine andfeline.

Recombinant HVT is useful as a vaccine against equine diseases whenforeign antigens from diseases or disease organisms are expressed in theHVT vector, including but not limited to: equine influenza, equineherpesvirus-1 and equine herpesvirus-4. Recombinant HVT is useful as avaccine against bovine diseases when foreign antigens from the followingdiseases or disease organisms are expressed in the HVT vector,including, but not limited to: bovine herpesvirus type 1, bovine viraldiarrhea virus, bovine respiratory syncytial virus, bovine parainfluenzavirus. Recombinant HVT is useful as a vaccine against swine diseaseswhen foreign antigens from the following diseases or disease organismsare expressed in the HVT vector, including but not limited to:pseudorabies virus, porcine reproductive and respiratory syndrome(PRRS/SIRS), hog cholera virus, swine influenza virus, swine parvovirus,swine rotavirus. Recombinant HVT is useful as a vaccine against felineor canine diseases when foreign antigens from the following diseases ordisease organisms are expressed in the HVT vector, including but notlimited to feline herpesvirus, feline leukemia virus, felineimmunodeficiency virus and Dirofilaria immitis (heartworm). Diseasecausing microorganisms in dogs include, but are not limited to canineherpesvirus, canine distemper, canine adenovirus type 1 (hepatitis),adenovirus type 2 (respiratory disease), parainfluenza, Leptospiracanicola, icterohemorragia, parvovirus, coronavirus, Borreliaburgdorferi, canine herpesvirus, Bordetella bronchiseptica, Dirofilariaimmitis (heartworm) and rabies virus.

EXAMPLE 22

Human Vaccines Using Recombinant Herpesvirus of Turkeys as a Vector

Recombinant herpesvirus of turkeys (HVT) is useful as a vaccine againsthuman diseases. For example, human influenza is a rapidly evolving viruswhose neutralizing viral epitopes are rapidly changing. A usefulrecombinant HVT vaccine is one in which the influenza neutralizingepitopes are quickly changed to protect against new strains ofinfluenza. Human influenza HA and NA genes are cloned using polymerasechain reaction into the recombinant HVT. Recombinant HVT is useful as avaccine against other human diseases when foreign antigens from thefollowing diseases or disease organisms are expressed in the HVT vector:hepatitis B virus surface and core antigens, hepatitis C virus, humanimmunodeficiency virus, herpes simplex virus-1, herpes simplex virus-2,human cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, humanherpesvirus-6, human herpesvirus-7, human influenza, measles virus,hantaan virus, pneumonia virus, rhinovirus, poliovirus, humanrespiratory syncytial virus, retrovirus, human T-cell leukemia virus,rabies virus, mumps virus, malaria (Plasmodium falciparum), Bordetellapertussis, Diptheria, Rickettsia prowazekii, Borrelia bergdorferi,Tetanus toxoid, malignant tumor antigens,

Recombinant HVT expressing human cytokines is combined with HVTexpressing genes for human disease antigens to enhance immune response.Additional cytokines, including, but not limited to,transforming growthfactor beta, epidermal growth factor family, fibroblast growth factors,hepatocyte growth factor, insulin-like growth factors, B-nerve growthfactor, platelet-derived growth factor, vascular endothelial growthfactor, interleukin 1, IL-1 receptor antagonist, interleukin 2,interleukin 3, interleukin 4, interleukin 5, interleukin 6, IL-6 solublereceptor, interleukin 7, interleukin 8, interleukin 9, interleukin 10,interleukin 11, interleukin 12, interleukin 13, angiogenin, chemokines,colony stimulating factors, granulocyte-macrophage colony stimulatingfactors, erythropoietin, interferon, interferon gamma, leukemiainhibitory factor, oncostatin M, pleiotrophin, secretory leukocyteprotease inhibitor, stem cell factor, tumor necrosis factors, andsoluble TNF receptors from human and other animals are expressed in HVTand have immune stimulating effects.

EXAMPLE 23

Improved Production of a Recombinant Herpesvirus of Turkeys Vaccine

Cytokines, such as interferons and interleukins, inhibit the replicationof viruses in cell culture and in the animal. Inhibition of theproduction of cellular interferon or interleukin improves the growth ofrecombinant HVT in cell culture. Chicken interferon (cIFN) expressedfrom a recombinant swinepox vector was added to chick embryo fibroblast(CEF) cell cultures and infected with S-HVT-012 which expressesβ-galactosidase. cIFN added to the cell culture media reduced both theexpression of β-galactosidase and S-HVT-012 titer in a dose dependentmanner. This result indicates that growth of HVT is limited by exogenousaddition of chicken interferon. Several strategies are utilized toimprove growth of HVT in CEF cells by removing or inactivating chickeninterferon activity in the CEF cells.

In one embodiment, a chicken interferon neutralizing antibody is addedto the culture medium to inhibit the chicken interferon activity andimprove the growth of recombinant HVT in CEF cell culture. The anti-cIFNantibody is derived from mouse or rabbit sera of animals injected withchicken interferon protein, preferably the cIFN is from a recombinantswinepox virus expressing chicken interferon.

Poxviruses secrete cytokine-inhibiting proteins as an immune evasionstrategy. One type of poxvirus immune evasion mechanism involvespoxvirus soluble receptors for interleukins, interferon, or tumornecrosis factors which inactive the cytokines and allow viralreplication (60). In an embodiment of the invention, fowlpox virus isuseful as a source of chicken interferon-inhibiting proteins and otherimmune evasion proteins. Conditioned media from FPV infected CEF cellcultures is added to the HVT infected CEF cells to inhibit interferonactivity and increase the HVT titer. In a further embodiment, therecombinant chicken interferon inhibiting protein or another poxvirusimmune evasion protein is expressed in a vector in combination with anHVT vaccine composition to increase the HVT titer.

Chicken embryo fibroblast cells have been engineered to express foreigngenes (61). in a further embodiment, an interferon-negative CEF cellline is constructed by the introduction of a vector expressing a geneencoding antisense RNA for chicken interferon into the CEF cell line.Recombinant HVT grown in an interferon-negative CEF cell linedemonstrate improved virus titers compared to HVT grown in an interferonproducing CEF cell line. In a further embodiment, a chickenmyelomonocytic growth factor (cMGF)-positive CEF cell line isconstructed by the introduction of a vector expressing the cMGF geneinto the CEF cells. Recombinant HVT grown in a cMGF-positive CEF cellline demonstrates improved virus titers compared to HVT grown in a cMGFnegative CEF cell line.

Recombinant HVT of the present invention is useful as a vaccine againstMarek's disease and against other diseases as outlined in previousexamples. An increased efficiency in growth of recombinant HVT in CEFcells is useful in production of the vaccine.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 60                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3350 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 129..2522                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1:                        - - GGATACGATC GGTCTGACCC GGGGGAGTCA CCCGGGGACA GCCGTCAAGG CC -            #TTGTTCCA     60                                                                 - - GGATAGAACT CCTCCTTCTA CAACGCTATC ATTGATGGTC AGTAGAGATC AG -            #ACAAACGA    120                                                                 - - TCGCAGCG ATG ACA AAC CTG CAA GAT CAA ACC CAA - #CAG ATT GTT CCG        TTC     170                                                                              Met Thr Asn Leu Gln A - #sp Gln Thr Gln Gln Ile Val Pro Phe                    1       - #        5          - #        10                         - - ATA CGG AGC CTT CTG ATG CCA ACA ACC GGA CC - #G GCG TCC ATT CCG GAG          218                                                                       Ile Arg Ser Leu Leu Met Pro Thr Thr Gly Pr - #o Ala Ser Ile Pro Glu            15                 - # 20                 - # 25                 - # 30       - - ACA CCC TGG AGA AGC ACA CTC TCA GGT CAG AG - #A CTG ACC TAC AAT TTG          266                                                                       Thr Pro Trp Arg Ser Thr Leu Ser Gly Gln Ar - #g Leu Thr Tyr Asn Leu                            35 - #                 40 - #                 45              - - ACT GTG GGG GAC ACA GGG TCA GGG CTA ATT GT - #C TTT TTC CCT GGA TTC          314                                                                       Thr Val Gly Asp Thr Gly Ser Gly Leu Ile Va - #l Phe Phe Pro Gly Phe                        50     - #             55     - #             60                  - - CCT GGC TCA ATT GTG GGT GCT CAC TAC ACA CT - #G CAG AGC AAT GGG AAC          362                                                                       Pro Gly Ser Ile Val Gly Ala His Tyr Thr Le - #u Gln Ser Asn Gly Asn                    65         - #         70         - #         75                      - - TAC AAG TTC GAT CGG ATG CTC CTG ACT GCC CA - #G AAC CTA CCG GCC AGT          410                                                                       Tyr Lys Phe Asp Arg Met Leu Leu Thr Ala Gl - #n Asn Leu Pro Ala Ser                80             - #     85             - #     90                          - - TAC AAC TAC TGC AGG CTA GTG AGT CGG AGT CT - #C ACA GTG AGG TCA AGC          458                                                                       Tyr Asn Tyr Cys Arg Leu Val Ser Arg Ser Le - #u Thr Val Arg Ser Ser            95                 - #100                 - #105                 - #110       - - ACA CTT CCT GGT GGC GTT TAT GCA CTA AAC GG - #C ACC ATA AAC GCC GTG          506                                                                       Thr Leu Pro Gly Gly Val Tyr Ala Leu Asn Gl - #y Thr Ile Asn Ala Val                           115  - #               120  - #               125              - - ACC TTC CAA GGA AGC CTG AGT GAA CTG ACA GA - #T GTT AGC TAC AAT GGG          554                                                                       Thr Phe Gln Gly Ser Leu Ser Glu Leu Thr As - #p Val Ser Tyr Asn Gly                       130      - #           135      - #           140                  - - TTG ATG TCT GCA ACA GCC AAC ATC AAC GAC AA - #A ATT GGG AAC GTC CTA          602                                                                       Leu Met Ser Ala Thr Ala Asn Ile Asn Asp Ly - #s Ile Gly Asn Val Leu                   145          - #       150          - #       155                      - - GTA GGG GAA GGG GTC ACC GTC CTC AGC TTA CC - #C ACA TCA TAT GAT CTT          650                                                                       Val Gly Glu Gly Val Thr Val Leu Ser Leu Pr - #o Thr Ser Tyr Asp Leu               160              - #   165              - #   170                          - - GGG TAT GTG AGG CTT GGT GAC CCC ATT CCC GC - #A ATA GGG CTT GAC CCA          698                                                                       Gly Tyr Val Arg Leu Gly Asp Pro Ile Pro Al - #a Ile Gly Leu Asp Pro           175                 1 - #80                 1 - #85                 1 -      #90                                                                              - - AAA ATG GTA GCC ACA TGT GAC AGC AGT GAC AG - #G CCC AGA GTC TAC        ACC      746                                                                    Lys Met Val Ala Thr Cys Asp Ser Ser Asp Ar - #g Pro Arg Val Tyr Thr                          195  - #               200  - #               205              - - ATA ACT GCA GCC GAT GAT TAC CAA TTC TCA TC - #A CAG TAC CAA CCA GGT          794                                                                       Ile Thr Ala Ala Asp Asp Tyr Gln Phe Ser Se - #r Gln Tyr Gln Pro Gly                       210      - #           215      - #           220                  - - GGG GTA ACA ATC ACA CTG TTC TCA GCC AAC AT - #T GAT GCC ATC ACA AGC          842                                                                       Gly Val Thr Ile Thr Leu Phe Ser Ala Asn Il - #e Asp Ala Ile Thr Ser                   225          - #       230          - #       235                      - - CTC AGC GTT GGG GGA GAG CTC GTG TTT CGA AC - #A AGC GTC CAC GGC CTT          890                                                                       Leu Ser Val Gly Gly Glu Leu Val Phe Arg Th - #r Ser Val His Gly Leu               240              - #   245              - #   250                          - - GTA CTG GGC GCC ACC ATC TAC CTC ATA GGC TT - #T GAT GGG ACA ACG GTA          938                                                                       Val Leu Gly Ala Thr Ile Tyr Leu Ile Gly Ph - #e Asp Gly Thr Thr Val           255                 2 - #60                 2 - #65                 2 -      #70                                                                              - - ATC ACC AGG GCT GTG GCC GCA AAC ACT GGG CT - #G ACG ACC GGC ACC        GAC      986                                                                    Ile Thr Arg Ala Val Ala Ala Asn Thr Gly Le - #u Thr Thr Gly Thr Asp                          275  - #               280  - #               285              - - AAC CTT ATG CCA TTC AAT CTT GTG ATT CCA AC - #A AAC GAG ATA ACC CAG         1034                                                                       Asn Leu Met Pro Phe Asn Leu Val Ile Pro Th - #r Asn Glu Ile Thr Gln                       290      - #           295      - #           300                  - - CCA ATC ACA TCC ATC AAA CTG GAG ATA GTG AC - #C TCC AAA AGT GGT GGT         1082                                                                       Pro Ile Thr Ser Ile Lys Leu Glu Ile Val Th - #r Ser Lys Ser Gly Gly                   305          - #       310          - #       315                      - - CAG GCA GGG GAT CAG ATG TTA TGG TCG GCA AG - #A GGG AGC CTA GCA GTG         1130                                                                       Gln Ala Gly Asp Gln Met Leu Trp Ser Ala Ar - #g Gly Ser Leu Ala Val               320              - #   325              - #   330                          - - ACG ATC CAT GGT GGC AAC TAT CCA GGG GCC CT - #C CGT CCC GTC ACG CTA         1178                                                                       Thr Ile His Gly Gly Asn Tyr Pro Gly Ala Le - #u Arg Pro Val Thr Leu           335                 3 - #40                 3 - #45                 3 -      #50                                                                              - - GTG GCC TAC GAA AGA GTG GCA ACA GGA TCC GT - #C GTT ACG GTC GCT        GGG     1226                                                                    Val Ala Tyr Glu Arg Val Ala Thr Gly Ser Va - #l Val Thr Val Ala Gly                          355  - #               360  - #               365              - - GTG AGC AAC TTC GAG CTG ATC CCA AAT CCT GA - #A CTA GCA AAG AAC CTG         1274                                                                       Val Ser Asn Phe Glu Leu Ile Pro Asn Pro Gl - #u Leu Ala Lys Asn Leu                       370      - #           375      - #           380                  - - GTT ACA GAA TAC GGC CGA TTT GAC CCA GGA GC - #C ATG AAC TAC ACA AAA         1322                                                                       Val Thr Glu Tyr Gly Arg Phe Asp Pro Gly Al - #a Met Asn Tyr Thr Lys                   385          - #       390          - #       395                      - - TTG ATA CTG AGT GAG AGG GAC CGT CTT GGC AT - #C AAG ACC GTC TGG CCA         1370                                                                       Leu Ile Leu Ser Glu Arg Asp Arg Leu Gly Il - #e Lys Thr Val Trp Pro               400              - #   405              - #   410                          - - ACA AGG GAG TAC ACT GAC TTT CGT GAA TAC TT - #C ATG GAG GTG GCC GAC         1418                                                                       Thr Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Ph - #e Met Glu Val Ala Asp           415                 4 - #20                 4 - #25                 4 -      #30                                                                              - - CTC AAC TCT CCC CTG AAG ATT GCA GGA GCA TT - #C GGC TTC AAA GAC        ATA     1466                                                                    Leu Asn Ser Pro Leu Lys Ile Ala Gly Ala Ph - #e Gly Phe Lys Asp Ile                          435  - #               440  - #               445              - - ATC CGG GCC ATA AGG AGG ATA GCT GTG CCG GT - #G GTC TCC ACA TTG TTC         1514                                                                       Ile Arg Ala Ile Arg Arg Ile Ala Val Pro Va - #l Val Ser Thr Leu Phe                       450      - #           455      - #           460                  - - CCA CCT GCC GCT CCC CTA GCC CAT GCA ATT GG - #G GAA GGT GTA GAC TAC         1562                                                                       Pro Pro Ala Ala Pro Leu Ala His Ala Ile Gl - #y Glu Gly Val Asp Tyr                   465          - #       470          - #       475                      - - CTG CTG GGC GAT GAG GCA CAG GCT GCT TCA GG - #A ACT GCT CGA GCC GCG         1610                                                                       Leu Leu Gly Asp Glu Ala Gln Ala Ala Ser Gl - #y Thr Ala Arg Ala Ala               480              - #   485              - #   490                          - - TCA GGA AAA GCA AGA GCT GCC TCA GGC CGC AT - #A AGG CAG CTG ACT CTC         1658                                                                       Ser Gly Lys Ala Arg Ala Ala Ser Gly Arg Il - #e Arg Gln Leu Thr Leu           495                 5 - #00                 5 - #05                 5 -      #10                                                                              - - GCC GCC GAC AAG GGG TAC GAG GTA GTC GCG AA - #T CTA TTC CAG GTG        CCC     1706                                                                    Ala Ala Asp Lys Gly Tyr Glu Val Val Ala As - #n Leu Phe Gln Val Pro                          515  - #               520  - #               525              - - CAG AAT CCC GTA GTC GAC GGG ATT CTT GCT TC - #A CCT GGG GTA CTC CGC         1754                                                                       Gln Asn Pro Val Val Asp Gly Ile Leu Ala Se - #r Pro Gly Val Leu Arg                       530      - #           535      - #           540                  - - GGT GCA CAC AAC CTC GAC TGC GTG TTA AGA GA - #G GGT GCC ACG CTA TTC         1802                                                                       Gly Ala His Asn Leu Asp Cys Val Leu Arg Gl - #u Gly Ala Thr Leu Phe                   545          - #       550          - #       555                      - - CCT GTG GTT ATT ACG ACA GTG GAA GAC GCC AT - #G ACA CCC AAA GCA TTG         1850                                                                       Pro Val Val Ile Thr Thr Val Glu Asp Ala Me - #t Thr Pro Lys Ala Leu               560              - #   565              - #   570                          - - AAC AGC AAA ATG TTT GCT GTC ATT GAA GGC GT - #G CGA GAA GAC CTC CAA         1898                                                                       Asn Ser Lys Met Phe Ala Val Ile Glu Gly Va - #l Arg Glu Asp Leu Gln           575                 5 - #80                 5 - #85                 5 -      #90                                                                              - - CCT CCA TCT CAA AGA GGA TCC TTC ATA CGA AC - #T CTC TCT GGA CAC        AGA     1946                                                                    Pro Pro Ser Gln Arg Gly Ser Phe Ile Arg Th - #r Leu Ser Gly His Arg                          595  - #               600  - #               605              - - GTC TAT GGA TAT GCT CCA GAT GGG GTA CTT CC - #A CTG GAG ACT GGG AGA         1994                                                                       Val Tyr Gly Tyr Ala Pro Asp Gly Val Leu Pr - #o Leu Glu Thr Gly Arg                       610      - #           615      - #           620                  - - GAC TAC ACC GTT GTC CCA ATA GAT GAT GTC TG - #G GAC GAC AGC ATT ATG         2042                                                                       Asp Tyr Thr Val Val Pro Ile Asp Asp Val Tr - #p Asp Asp Ser Ile Met                   625          - #       630          - #       635                      - - CTG TCC AAA GAT CCC ATA CCT CCT ATT GTG GG - #A AAC AGT GGA AAT CTA         2090                                                                       Leu Ser Lys Asp Pro Ile Pro Pro Ile Val Gl - #y Asn Ser Gly Asn Leu               640              - #   645              - #   650                          - - GCC ATA GCT TAC ATG GAT GTG TTT CGA CCC AA - #A GTC CCA ATC CAT GTG         2138                                                                       Ala Ile Ala Tyr Met Asp Val Phe Arg Pro Ly - #s Val Pro Ile His Val            655                 - #660                 - #665                 -         #670                                                                             - - GCT ATG ACG GGA GCC CTC AAT GCT TGT GGC GA - #G ATT GAG AAA GTA        AGC     2186                                                                    Ala Met Thr Gly Ala Leu Asn Ala Cys Gly Gl - #u Ile Glu Lys Val Ser                          675  - #               680  - #               685              - - TTT AGA AGC ACC AAG CTC GCC ACT GCA CAC CG - #A CTT GGC CTT AAG TTG         2234                                                                       Phe Arg Ser Thr Lys Leu Ala Thr Ala His Ar - #g Leu Gly Leu Lys Leu                       690      - #           695      - #           700                  - - GCT GGT CCC GGA GCA TTC GAT GTA AAC ACC GG - #G CCC AAC TGG GCA ACG         2282                                                                       Ala Gly Pro Gly Ala Phe Asp Val Asn Thr Gl - #y Pro Asn Trp Ala Thr                   705          - #       710          - #       715                      - - TTC ATC AAA CGT TTC CCT CAC AAT CCA CGC GA - #C TGG GAC AGG CTC CCC         2330                                                                       Phe Ile Lys Arg Phe Pro His Asn Pro Arg As - #p Trp Asp Arg Leu Pro               720              - #   725              - #   730                          - - TAC CTC AAC CTA CCA TAC CTT CCA CCC AAT GC - #A GGA CGC CAG TAC CAC         2378                                                                       Tyr Leu Asn Leu Pro Tyr Leu Pro Pro Asn Al - #a Gly Arg Gln Tyr His           735                 7 - #40                 7 - #45                 7 -      #50                                                                              - - CTT GCC ATG GCT GCA TCA GAG TTC AAG AGA CC - #C CGA ACT CGA GAG        TGC     2426                                                                    Leu Ala Met Ala Ala Ser Glu Phe Lys Arg Pr - #o Arg Thr Arg Glu Cys                          755  - #               760  - #               765              - - CGT CAG AGC AAT GGA AGC AGC AGC CAA CGT GG - #A CCC ACT ATT CCA ATC         2474                                                                       Arg Gln Ser Asn Gly Ser Ser Ser Gln Arg Gl - #y Pro Thr Ile Pro Ile                       770      - #           775      - #           780                  - - TGC ACT CAG TGT GTT CAT GTG GCT GGA AGA GA - #A TGG GAT TGT GAC TGA         2522                                                                       Cys Thr Gln Cys Val His Val Ala Gly Arg Gl - #u Trp Asp Cys Asp                       785          - #       790          - #       795                      - - CATGGCCAAC TTCGCACTCA GCGACCCGAA CGCCCATCGG ATGCGAAATT TT -             #TTTGCAAA   2582                                                                 - - CGACCACAAG CAGGCAGCAA GTCGCAAAGG GCCAAGTACG GGACAGCAGG CT -            #ACGGAGTG   2642                                                                 - - GAGGCTCGGG GCCCCCACAC CAGAGGAAGC ACAGAGGGAA AAAGACACAC GG -            #ATCTCAAA   2702                                                                 - - GAAGATGGAG ACCATGGGCA TCTACTTTGC AACACCAGAA TGGGTAGCAC TC -            #AATGGGCA   2762                                                                 - - CCGAGGGCCA AGCCCCGGCC AGCTAAAGTA CGGGCAGAAC ACACGAGAAA TA -            #CGGACCCA   2822                                                                 - - AACGAGGACT ATCTAGACTA CGTGCATGCA GAGAAGAGCC GGTTGGCATC AG -            #AAGAACAA   2882                                                                 - - ATCCTAAGGG CAGCTACGTC AGATCTACGG GGCTCCAGGA CAGGCAGAGC AC -            #CCCAAGCT   2942                                                                 - - TTCATAGACG AAGTTGCCAA AGTCTATGAA ATCAACCATG GACGTGGCCC AA -            #ACCAAGAA   3002                                                                 - - CAGATGAAAG ATCTGCTCTT GACTGCGATG GAGATGAAGC ATCGCAATCC CA -            #GGCGGGCT   3062                                                                 - - CTACCAAAGC CCAAGCCAAA ACCCAATGCT CCAACACAGA GACCCCCTGG TC -            #GGCTGGGG   3122                                                                 - - CTGGATCAGG ACCGTCTCTG ATGAGGACCT TGAGTGAGGC TCCTGGGAGT CT -            #CCCGACAA   3182                                                                 - - CACCCGCGCA GGTGTGGACA CAATTCGGCC TTACAACATC CCAAATTGGA TC -            #CGTTCGCG   3242                                                                 - - GGTCCCCAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA -            #AAAAAAAA   3302                                                                 - - AAGTACCTTC TGAGGCGGAA AGAACCAGCC GGATCCCTCG AGGGATCC  - #                  3350                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 797 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:2:                     - - Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Il - #e Val Pro Phe Ile Arg        1               5 - #                 10 - #                 15              - - Ser Leu Leu Met Pro Thr Thr Gly Pro Ala Se - #r Ile Pro Glu Thr Pro                   20     - #             25     - #             30                  - - Trp Arg Ser Thr Leu Ser Gly Gln Arg Leu Th - #r Tyr Asn Leu Thr Val               35         - #         40         - #         45                      - - Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Ph - #e Pro Gly Phe Pro Gly           50             - #     55             - #     60                          - - Ser Ile Val Gly Ala His Tyr Thr Leu Gln Se - #r Asn Gly Asn Tyr Lys       65                 - # 70                 - # 75                 - # 80       - - Phe Asp Arg Met Leu Leu Thr Ala Gln Asn Le - #u Pro Ala Ser Tyr Asn                       85 - #                 90 - #                 95              - - Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Va - #l Arg Ser Ser Thr Leu                  100      - #           105      - #           110                  - - Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Il - #e Asn Ala Val Thr Phe              115          - #       120          - #       125                      - - Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Se - #r Tyr Asn Gly Leu Met          130              - #   135              - #   140                          - - Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gl - #y Asn Val Leu Val Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Gly Val Thr Val Leu Ser Leu Pro Thr Se - #r Tyr Asp Leu Gly        Tyr                                                                                             165  - #               170  - #               175             - - Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gl - #y Leu Asp Pro Lys Met                  180      - #           185      - #           190                  - - Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Ar - #g Val Tyr Thr Ile Thr              195          - #       200          - #       205                      - - Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Ty - #r Gln Pro Gly Gly Val          210              - #   215              - #   220                          - - Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Al - #a Ile Thr Ser Leu Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Gly Gly Glu Leu Val Phe Arg Thr Ser Va - #l His Gly Leu Val        Leu                                                                                             245  - #               250  - #               255             - - Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gl - #y Thr Thr Val Ile Thr                  260      - #           265      - #           270                  - - Arg Ala Val Ala Ala Asn Thr Gly Leu Thr Th - #r Gly Thr Asp Asn Leu              275          - #       280          - #       285                      - - Met Pro Phe Asn Leu Val Ile Pro Thr Asn Gl - #u Ile Thr Gln Pro Ile          290              - #   295              - #   300                          - - Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Ly - #s Ser Gly Gly Gln Ala      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Asp Gln Met Leu Trp Ser Ala Arg Gly Se - #r Leu Ala Val Thr        Ile                                                                                             325  - #               330  - #               335             - - His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pr - #o Val Thr Leu Val Ala                  340      - #           345      - #           350                  - - Tyr Glu Arg Val Ala Thr Gly Ser Val Val Th - #r Val Ala Gly Val Ser              355          - #       360          - #       365                      - - Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Al - #a Lys Asn Leu Val Thr          370              - #   375              - #   380                          - - Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met As - #n Tyr Thr Lys Leu Ile      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Th - #r Val Trp Pro Thr        Arg                                                                                             405  - #               410  - #               415             - - Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Gl - #u Val Ala Asp Leu Asn                  420      - #           425      - #           430                  - - Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Ph - #e Lys Asp Ile Ile Arg              435          - #       440          - #       445                      - - Ala Ile Arg Arg Ile Ala Val Pro Val Val Se - #r Thr Leu Phe Pro Pro          450              - #   455              - #   460                          - - Ala Ala Pro Leu Ala His Ala Ile Gly Glu Gl - #y Val Asp Tyr Leu Leu      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Gly Asp Glu Ala Gln Ala Ala Ser Gly Thr Al - #a Arg Ala Ala Ser        Gly                                                                                             485  - #               490  - #               495             - - Lys Ala Arg Ala Ala Ser Gly Arg Ile Arg Gl - #n Leu Thr Leu Ala Ala                  500      - #           505      - #           510                  - - Asp Lys Gly Tyr Glu Val Val Ala Asn Leu Ph - #e Gln Val Pro Gln Asn              515          - #       520          - #       525                      - - Pro Val Val Asp Gly Ile Leu Ala Ser Pro Gl - #y Val Leu Arg Gly Ala          530              - #   535              - #   540                          - - His Asn Leu Asp Cys Val Leu Arg Glu Gly Al - #a Thr Leu Phe Pro Val      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Val Ile Thr Thr Val Glu Asp Ala Met Thr Pr - #o Lys Ala Leu Asn        Ser                                                                                             565  - #               570  - #               575             - - Lys Met Phe Ala Val Ile Glu Gly Val Arg Gl - #u Asp Leu Gln Pro Pro                  580      - #           585      - #           590                  - - Ser Gln Arg Gly Ser Phe Ile Arg Thr Leu Se - #r Gly His Arg Val Tyr              595          - #       600          - #       605                      - - Gly Tyr Ala Pro Asp Gly Val Leu Pro Leu Gl - #u Thr Gly Arg Asp Tyr          610              - #   615              - #   620                          - - Thr Val Val Pro Ile Asp Asp Val Trp Asp As - #p Ser Ile Met Leu Ser      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Lys Asp Pro Ile Pro Pro Ile Val Gly Asn Se - #r Gly Asn Leu Ala        Ile                                                                                             645  - #               650  - #               655             - - Ala Tyr Met Asp Val Phe Arg Pro Lys Val Pr - #o Ile His Val Ala Met                  660      - #           665      - #           670                  - - Thr Gly Ala Leu Asn Ala Cys Gly Glu Ile Gl - #u Lys Val Ser Phe Arg              675          - #       680          - #       685                      - - Ser Thr Lys Leu Ala Thr Ala His Arg Leu Gl - #y Leu Lys Leu Ala Gly          690              - #   695              - #   700                          - - Pro Gly Ala Phe Asp Val Asn Thr Gly Pro As - #n Trp Ala Thr Phe Ile      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Lys Arg Phe Pro His Asn Pro Arg Asp Trp As - #p Arg Leu Pro Tyr        Leu                                                                                             725  - #               730  - #               735             - - Asn Leu Pro Tyr Leu Pro Pro Asn Ala Gly Ar - #g Gln Tyr His Leu Ala                  740      - #           745      - #           750                  - - Met Ala Ala Ser Glu Phe Lys Arg Pro Arg Th - #r Arg Glu Cys Arg Gln              755          - #       760          - #       765                      - - Ser Asn Gly Ser Ser Ser Gln Arg Gly Pro Th - #r Ile Pro Ile Cys Thr          770              - #   775              - #   780                          - - Gln Cys Val His Val Ala Gly Arg Glu Trp As - #p Cys Asp                  785                 7 - #90                 7 - #95                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5426 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 73..1182                                                        (D) OTHER INFORMATION: - #/product= "HVT UL42"                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1306..2574                                                      (D) OTHER INFORMATION: - #/product= "HVT UL43"                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 2790..4259                                                      (D) OTHER INFORMATION: - #/product= "HVT gA"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 4701..5339                                                      (D) OTHER INFORMATION: - #/product= "HVT UL45"                       - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:3:                        - - GGATCCGAGC TTCTACTATA CAACGCGGAC GATAATTTTG TCCACCCCAT CG -             #GTGTTCGA     60                                                                 - - GAAAGGGTTT TT ATG ATG GCA GGA ATA ACT GTC GCA - # TGT GAC CAC ACT            108                                                                                    Met Met Al - #a Gly Ile Thr Val Ala Cys Asp His Thr                              1 - #              5    - #              10                   - - GCA GGA GAG GCT CAT ACA CCC GAG GAT ATG CA - #A AAG AAA TGG AGG ATT          156                                                                       Ala Gly Glu Ala His Thr Pro Glu Asp Met Gl - #n Lys Lys Trp Arg Ile                    15         - #         20         - #         25                      - - ATA TTG GCA GGG GAA AAA TTC ATG ACT ATA TC - #G GCA TCG TTG AAA TCG          204                                                                       Ile Leu Ala Gly Glu Lys Phe Met Thr Ile Se - #r Ala Ser Leu Lys Ser                30             - #     35             - #     40                          - - ATC GTC AGT TGT GTG AAA AAC CCC CTT CTC AC - #G TTT GGC GCA GAT GGG          252                                                                       Ile Val Ser Cys Val Lys Asn Pro Leu Leu Th - #r Phe Gly Ala Asp Gly            45                 - # 50                 - # 55                 - # 60       - - CTC ATT GTA CAA GGT ACT GTC TGC GGA CAG CG - #C ATT TTT GTT CCA ATC          300                                                                       Leu Ile Val Gln Gly Thr Val Cys Gly Gln Ar - #g Ile Phe Val Pro Ile                            65 - #                 70 - #                 75              - - GAC CGT GAT TCC TTC AGC GAA TAT GAA TGG CA - #T GGG CCA ACT GCG ATG          348                                                                       Asp Arg Asp Ser Phe Ser Glu Tyr Glu Trp Hi - #s Gly Pro Thr Ala Met                        80     - #             85     - #             90                  - - TTT CTA GCA TTA ACT GAT TCC AGA CGC ACT CT - #T TTA GAT GCA TTC AAA          396                                                                       Phe Leu Ala Leu Thr Asp Ser Arg Arg Thr Le - #u Leu Asp Ala Phe Lys                    95         - #        100         - #        105                      - - TGT GAA AAG AGA AGG GCA ATT GAC GTC TCC TT - #T ACC TTC GCG GGA GAG          444                                                                       Cys Glu Lys Arg Arg Ala Ile Asp Val Ser Ph - #e Thr Phe Ala Gly Glu               110              - #   115              - #   120                          - - CCT CCA TGT AGG CAT TTA ATC CAA GCC GTC AC - #A TAC ATG ACC GAC GGT          492                                                                       Pro Pro Cys Arg His Leu Ile Gln Ala Val Th - #r Tyr Met Thr Asp Gly           125                 1 - #30                 1 - #35                 1 -      #40                                                                              - - GGT TCA GTA TCG AAT ACA ATC ATT AAA TAT GA - #G CTC TGG AAT GCG        TCT      540                                                                    Gly Ser Val Ser Asn Thr Ile Ile Lys Tyr Gl - #u Leu Trp Asn Ala Ser                          145  - #               150  - #               155              - - ACA ATT TTC CCC CAA AAA ACT CCC GAT GTT AC - #C TTT TCT CTA AAC AAA          588                                                                       Thr Ile Phe Pro Gln Lys Thr Pro Asp Val Th - #r Phe Ser Leu Asn Lys                       160      - #           165      - #           170                  - - CAA CAA TTG AAC AAA ATA TTG GCC GTC GCT TC - #A AAA CTG CAA CAC GAA          636                                                                       Gln Gln Leu Asn Lys Ile Leu Ala Val Ala Se - #r Lys Leu Gln His Glu                   175          - #       180          - #       185                      - - GAA CTT GTA TTC TCT TTA AAA CCT GAA GGA GG - #G TTC TAC GTA GGA ACG          684                                                                       Glu Leu Val Phe Ser Leu Lys Pro Glu Gly Gl - #y Phe Tyr Val Gly Thr               190              - #   195              - #   200                          - - GTT TGT ACT GTT ATA AGT TTC GAA GTA GAT GG - #G ACT GCC ATG ACT CAG          732                                                                       Val Cys Thr Val Ile Ser Phe Glu Val Asp Gl - #y Thr Ala Met Thr Gln           205                 2 - #10                 2 - #15                 2 -      #20                                                                              - - TAT CCT TAC AAC CCT CCA ACC TCG GCT ACC CT - #A GCT CTC GTA GTA        GCA      780                                                                    Tyr Pro Tyr Asn Pro Pro Thr Ser Ala Thr Le - #u Ala Leu Val Val Ala                          225  - #               230  - #               235              - - TGC AGA AAG AAG AAG GCG AAT AAA AAC ACT AT - #T TTA ACG GCC TAT GGA          828                                                                       Cys Arg Lys Lys Lys Ala Asn Lys Asn Thr Il - #e Leu Thr Ala Tyr Gly                       240      - #           245      - #           250                  - - AGT GGT AAA CCC TTT TGT GTT GCA TTG GAA GA - #T ACT AGT GCA TTT AGA          876                                                                       Ser Gly Lys Pro Phe Cys Val Ala Leu Glu As - #p Thr Ser Ala Phe Arg                   255          - #       260          - #       265                      - - AAT ATC GTC AAT AAA ATC AAG GCG GGT ACG TC - #G GGA GTT GAT CTG GGG          924                                                                       Asn Ile Val Asn Lys Ile Lys Ala Gly Thr Se - #r Gly Val Asp Leu Gly               270              - #   275              - #   280                          - - TTT TAT ACA ACT TGC GAT CCG CCG ATG CTA TG - #T ATT CGC CCA CAC GCA          972                                                                       Phe Tyr Thr Thr Cys Asp Pro Pro Met Leu Cy - #s Ile Arg Pro His Ala           285                 2 - #90                 2 - #95                 3 -      #00                                                                              - - TTT GGA AGT CCT ACC GCA TTC CTG TTT TGT AA - #C ACA GAC TGT ATG        ACA     1020                                                                    Phe Gly Ser Pro Thr Ala Phe Leu Phe Cys As - #n Thr Asp Cys Met Thr                          305  - #               310  - #               315              - - ATA TAT GAA CTG GAA GAA GTA AGC GCC GTT GA - #T GGT GCA ATC CGA GCA         1068                                                                       Ile Tyr Glu Leu Glu Glu Val Ser Ala Val As - #p Gly Ala Ile Arg Ala                       320      - #           325      - #           330                  - - AAA CGC ATC AAC GAA TAT TTC CCA ACA GTA TC - #G CAG GCT ACT TCC AAG         1116                                                                       Lys Arg Ile Asn Glu Tyr Phe Pro Thr Val Se - #r Gln Ala Thr Ser Lys                   335          - #       340          - #       345                      - - AAG AGA AAA CAG TCG CCG CCC CCT ATC GAA AG - #A GAA AGG AAA ACC ACC         1164                                                                       Lys Arg Lys Gln Ser Pro Pro Pro Ile Glu Ar - #g Glu Arg Lys Thr Thr               350              - #   355              - #   360                          - - AGA GCG GAT ACC CAA TAAAATGCCA GACAAACCCG GCATCCTGG - #T TAGAGGGCAG         1219                                                                       Arg Ala Asp Thr Gln                                                           365                 3 - #70                                                    - - GTGGGCTGGG CCAACCTTCA CGGGCGTCCG ACAGATCGGT GACACTCATA CG -             #TTAACTAA   1279                                                                 - - ACGCCGGCAG CTTTGCAGAA GAAAAT ATG CCT TCC GGA GCC - #AGC TCG AGT        CCT    1332                                                                                       - #           Met Pro Ser Gly - #Ala Ser Ser Ser Pro                         - #             1     - #          5                         - - CCA CCA GCT TAT ACA TCT GCA GCT CCG CTT GA - #G ACT TAT AAC AGC TGG         1380                                                                       Pro Pro Ala Tyr Thr Ser Ala Ala Pro Leu Gl - #u Thr Tyr Asn Ser Trp            10                 - # 15                 - # 20                 - # 25       - - CTA AGT GCC TTT TCA TGC GCA TAT CCC CAA TG - #C ACT GCG GGA AGA GGA         1428                                                                       Leu Ser Ala Phe Ser Cys Ala Tyr Pro Gln Cy - #s Thr Ala Gly Arg Gly                            30 - #                 35 - #                 40              - - CAT CGA CAA AAT GGC AAG AAG TGT ATA CGG TG - #T ATA GTG ATC AGT GTA         1476                                                                       His Arg Gln Asn Gly Lys Lys Cys Ile Arg Cy - #s Ile Val Ile Ser Val                        45     - #             50     - #             55                  - - TGT TCC TTA GTG TGC ATC GCT GCA CAT TTA GC - #T GTT ACC GTG TCG GGA         1524                                                                       Cys Ser Leu Val Cys Ile Ala Ala His Leu Al - #a Val Thr Val Ser Gly                    60         - #         65         - #         70                      - - GTG GCA TTA ATT CCG CTT ATC GAT CAA AAC AG - #A GCT TAC GGA AAC TGT         1572                                                                       Val Ala Leu Ile Pro Leu Ile Asp Gln Asn Ar - #g Ala Tyr Gly Asn Cys                75             - #     80             - #     85                          - - ACG GTA TGT GTA ATT GCC GGA TTC ATC GCT AC - #G TTT GCT GCA CGA CTT         1620                                                                       Thr Val Cys Val Ile Ala Gly Phe Ile Ala Th - #r Phe Ala Ala Arg Leu            90                 - # 95                 - #100                 - #105       - - ACG ATA AGA CTT TCG GAA ACG CTT ATG CTA GT - #G GGC AAG CCG GCG CAG         1668                                                                       Thr Ile Arg Leu Ser Glu Thr Leu Met Leu Va - #l Gly Lys Pro Ala Gln                           110  - #               115  - #               120              - - TTT ATA TTT GCT ATA ATC GCT TCC GTT GCG GA - #A ACA CTG ATC AAT AAC         1716                                                                       Phe Ile Phe Ala Ile Ile Ala Ser Val Ala Gl - #u Thr Leu Ile Asn Asn                       125      - #           130      - #           135                  - - GAG GCG CTT GCC ATC AGT AAT ACT ACT TAC AA - #A ACT GCA TTG CGA ATA         1764                                                                       Glu Ala Leu Ala Ile Ser Asn Thr Thr Tyr Ly - #s Thr Ala Leu Arg Ile                   140          - #       145          - #       150                      - - ATC GAA GTA ACA TCT TTG GCG TGT TTT GTT AT - #G CTC GGG GCA ATA ATT         1812                                                                       Ile Glu Val Thr Ser Leu Ala Cys Phe Val Me - #t Leu Gly Ala Ile Ile               155              - #   160              - #   165                          - - ACA TCC CAC AAC TAT GTC TGC ATT TCA ACG GC - #A GGG GAC TTG ACT TGG         1860                                                                       Thr Ser His Asn Tyr Val Cys Ile Ser Thr Al - #a Gly Asp Leu Thr Trp           170                 1 - #75                 1 - #80                 1 -      #85                                                                              - - AAG GGC GGG ATT TTT CAT GCT TAC CAC GGA AC - #A TTA CTC GGT ATA        ACA     1908                                                                    Lys Gly Gly Ile Phe His Ala Tyr His Gly Th - #r Leu Leu Gly Ile Thr                          190  - #               195  - #               200              - - ATA CCA AAC ATA CAC CCA ATC CCT CTC GCG GG - #G TTT CTT GCA GTC TAT         1956                                                                       Ile Pro Asn Ile His Pro Ile Pro Leu Ala Gl - #y Phe Leu Ala Val Tyr                       205      - #           210      - #           215                  - - ACA ATA TTG GCT ATA AAT ATC GCT AGA GAT GC - #A AGC GCT ACA TTA TTA         2004                                                                       Thr Ile Leu Ala Ile Asn Ile Ala Arg Asp Al - #a Ser Ala Thr Leu Leu                   220          - #       225          - #       230                      - - TCC ACT TGC TAT TAT CGC AAT TGC CGC GAG AG - #G ACT ATA CTT CGC CCT         2052                                                                       Ser Thr Cys Tyr Tyr Arg Asn Cys Arg Glu Ar - #g Thr Ile Leu Arg Pro               235              - #   240              - #   245                          - - TCT CGT CTC GGA CAT GGT TAC ACA ATC CCT TC - #T CCC GGT GCC GAT ATG         2100                                                                       Ser Arg Leu Gly His Gly Tyr Thr Ile Pro Se - #r Pro Gly Ala Asp Met           250                 2 - #55                 2 - #60                 2 -      #65                                                                              - - CTT TAT GAA GAA GAC GTA TAT AGT TTT GAC GC - #A GCT AAA GGC CAT        TAT     2148                                                                    Leu Tyr Glu Glu Asp Val Tyr Ser Phe Asp Al - #a Ala Lys Gly His Tyr                          270  - #               275  - #               280              - - TCG TCA ATA TTT CTA TGT TAT GCC ATG GGG CT - #T ACA ACA CCG CTG ATT         2196                                                                       Ser Ser Ile Phe Leu Cys Tyr Ala Met Gly Le - #u Thr Thr Pro Leu Ile                       285      - #           290      - #           295                  - - ATT GCG CTC CAT AAA TAT ATG GCG GGC ATT AA - #A AAT TCG TCA GAT TGG         2244                                                                       Ile Ala Leu His Lys Tyr Met Ala Gly Ile Ly - #s Asn Ser Ser Asp Trp                   300          - #       305          - #       310                      - - ACT GCT ACA TTA CAA GGC ATG TAC GGG CTT GT - #C TTG GGA TCG CTA TCG         2292                                                                       Thr Ala Thr Leu Gln Gly Met Tyr Gly Leu Va - #l Leu Gly Ser Leu Ser               315              - #   320              - #   325                          - - TCA CTA TGT ATT CCA TCC AGC AAC AAC GAT GC - #C CTA ATT CGT CCC ATT         2340                                                                       Ser Leu Cys Ile Pro Ser Ser Asn Asn Asp Al - #a Leu Ile Arg Pro Ile           330                 3 - #35                 3 - #40                 3 -      #45                                                                              - - CAA ATT TTG ATA TTG ATA ATC GGT GCA CTG GC - #C ATT GCA TTG GCT        GGA     2388                                                                    Gln Ile Leu Ile Leu Ile Ile Gly Ala Leu Al - #a Ile Ala Leu Ala Gly                          350  - #               355  - #               360              - - TGT GGT CAA ATT ATA GGG CCT ACA TTA TTT GC - #C GCG AGT TCG GCT GCG         2436                                                                       Cys Gly Gln Ile Ile Gly Pro Thr Leu Phe Al - #a Ala Ser Ser Ala Ala                       365      - #           370      - #           375                  - - ATG TCA TGT TTT ACA TGT ATC AAT ATT CGC GC - #T ACT AAT AAG GGT GTC         2484                                                                       Met Ser Cys Phe Thr Cys Ile Asn Ile Arg Al - #a Thr Asn Lys Gly Val                   380          - #       385          - #       390                      - - AAC AAA TTG GCA GCA GCC AGT GTC GTG AAA TC - #T GTA CTG GGC TTC ATT         2532                                                                       Asn Lys Leu Ala Ala Ala Ser Val Val Lys Se - #r Val Leu Gly Phe Ile               395              - #   400              - #   405                          - - ATT TCC GGG ATG CTT ACT TGC GTG CTA TTA CC - #A CTA TCG TGATAGATCG          2581                                                                       Ile Ser Gly Met Leu Thr Cys Val Leu Leu Pr - #o Leu Ser                       410                 4 - #15                 4 - #20                            - - TCGGTCTGCG CATCGCCCAT GCTGGCGGAA CGCTCTTTCG AACCGTGAAT AA -             #AACTTTGT   2641                                                                 - - ATCTACTAAA CAATAACTTT GTGTTTTATT GAGCGGTCGA AAACAATGAG GA -            #GCTGCAAT   2701                                                                 - - TTAAAGCTAA CCGCATACGC CGGGCGGGTA AAGACCATTT TATACCATAT TA -            #CGCATCTA   2761                                                                 - - TCGAAACTTG TTCGAGAACC GCAAGTAT ATG GTT TCC AAC ATG - #CGC GTT CTA          2813                                                                                         - #             Met Val Ser - #Asn Met Arg Val Leu                            - #               1   - #            5                       - - CGC GTA CTG CGC CTG ACG GGA TGG GTG GGC AT - #A TTT CTA GTT CTG TCT         2861                                                                       Arg Val Leu Arg Leu Thr Gly Trp Val Gly Il - #e Phe Leu Val Leu Ser                10             - #     15             - #     20                          - - TTA CAG CAA ACC TCT TGT GCC GGA TTG CCC CA - #T AAC GTC GAT ACC CAT         2909                                                                       Leu Gln Gln Thr Ser Cys Ala Gly Leu Pro Hi - #s Asn Val Asp Thr His            25                 - # 30                 - # 35                 - # 40       - - CAT ATC CTA ACT TTC AAC CCT TCT CCC ATT TC - #G GCC GAT GGC GTT CCT         2957                                                                       His Ile Leu Thr Phe Asn Pro Ser Pro Ile Se - #r Ala Asp Gly Val Pro                            45 - #                 50 - #                 55              - - TTG TCA GAG GTG CCC AAT TCG CCT ACG ACC GA - #A TTA TCT ACA ACT GTC         3005                                                                       Leu Ser Glu Val Pro Asn Ser Pro Thr Thr Gl - #u Leu Ser Thr Thr Val                        60     - #             65     - #             70                  - - GCC ACC AAG ACA GCT GTA CCG ACG ACT GAA AG - #C ACT AGT TCC TCC GAA         3053                                                                       Ala Thr Lys Thr Ala Val Pro Thr Thr Glu Se - #r Thr Ser Ser Ser Glu                    75         - #         80         - #         85                      - - GCG CAC CGC AAC TCT TCT CAC AAA ATA CCT GA - #T ATA ATC TGC GAC CGA         3101                                                                       Ala His Arg Asn Ser Ser His Lys Ile Pro As - #p Ile Ile Cys Asp Arg                90             - #     95             - #    100                          - - GAA GAA GTA TTC GTA TTC CTT AAC AAT ACA GG - #A AGA ATT TTG TGT GAC         3149                                                                       Glu Glu Val Phe Val Phe Leu Asn Asn Thr Gl - #y Arg Ile Leu Cys Asp            105                 - #110                 - #115                 -         #120                                                                             - - CTT ATA GTC GAC CCC CCT TCA GAC GAT GAA TG - #G TCC AAC TTC GCT        CTT     3197                                                                    Leu Ile Val Asp Pro Pro Ser Asp Asp Glu Tr - #p Ser Asn Phe Ala Leu                          125  - #               130  - #               135              - - GAC GTC ACG TTC AAT CCA ATC GAA TAC CAC GC - #C AAC GAA AAG AAT GTA         3245                                                                       Asp Val Thr Phe Asn Pro Ile Glu Tyr His Al - #a Asn Glu Lys Asn Val                       140      - #           145      - #           150                  - - GAG GTT GCC CGA GTG GCC GGT CTA TAC GGA GT - #A CCG GGG TCG GAT TAT         3293                                                                       Glu Val Ala Arg Val Ala Gly Leu Tyr Gly Va - #l Pro Gly Ser Asp Tyr                   155          - #       160          - #       165                      - - GCA TAC CCT AGG AAA TCG GAA TTA ATA TCC TC - #C ATT CGA CGG GAT CCC         3341                                                                       Ala Tyr Pro Arg Lys Ser Glu Leu Ile Ser Se - #r Ile Arg Arg Asp Pro               170              - #   175              - #   180                          - - CAG GGT TCT TTC TGG ACT AGT CCT ACA CCC CG - #T GGA AAT AAA TAT TTC         3389                                                                       Gln Gly Ser Phe Trp Thr Ser Pro Thr Pro Ar - #g Gly Asn Lys Tyr Phe           185                 1 - #90                 1 - #95                 2 -      #00                                                                              - - ATA TGG ATT AAT AAA ACA ATG CAC ACC ATG GG - #C GTG GAA GTT AGA        AAT     3437                                                                    Ile Trp Ile Asn Lys Thr Met His Thr Met Gl - #y Val Glu Val Arg Asn                          205  - #               210  - #               215              - - GTC GAC TAC AAA GAC AAC GGC TAC TTT CAA GT - #G ATA CTG CGT GAT AGA         3485                                                                       Val Asp Tyr Lys Asp Asn Gly Tyr Phe Gln Va - #l Ile Leu Arg Asp Arg                       220      - #           225      - #           230                  - - TTT AAT CGC CCA TTG GTA GAA AAA CAT ATT TA - #C ATG CGT GTG TGC CAA         3533                                                                       Phe Asn Arg Pro Leu Val Glu Lys His Ile Ty - #r Met Arg Val Cys Gln                   235          - #       240          - #       245                      - - CGA CCC GCA TCC GTG GAT GTA TTG GCC CCT CC - #A GTT CTC AGC GGA GAA         3581                                                                       Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pr - #o Val Leu Ser Gly Glu               250              - #   255              - #   260                          - - AAC TAC AAA GCA TCT TGC ATC GTT AGA CAT TT - #T TAT CCC CCG GGA TCT         3629                                                                       Asn Tyr Lys Ala Ser Cys Ile Val Arg His Ph - #e Tyr Pro Pro Gly Ser           265                 2 - #70                 2 - #75                 2 -      #80                                                                              - - GTC TAC GTA TCT TGG AGA CGT AAC GGA AAC AT - #T GCC ACA CCC CGC        AAG     3677                                                                    Val Tyr Val Ser Trp Arg Arg Asn Gly Asn Il - #e Ala Thr Pro Arg Lys                          285  - #               290  - #               295              - - GAC CGT GAC GGG AGT TTT TGG TGG TTC GAA TC - #T GGC CGC GGG GCC ACA         3725                                                                       Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Se - #r Gly Arg Gly Ala Thr                       300      - #           305      - #           310                  - - CTA GTA TCC ACA ATA ACC CTC GGA AAC TCT GG - #A CTC GAA TCT CCT CCA         3773                                                                       Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gl - #y Leu Glu Ser Pro Pro                   315          - #       320          - #       325                      - - AAG GTT TCC TGC TTG GTA GCG TGG AGG CAA GG - #C GAT ATG ATA AGC ACA         3821                                                                       Lys Val Ser Cys Leu Val Ala Trp Arg Gln Gl - #y Asp Met Ile Ser Thr               330              - #   335              - #   340                          - - TCG AAT GCT ACA GCT GTA CCG ACG GTA TAT TA - #T CAC CCC CGT ATC TCT         3869                                                                       Ser Asn Ala Thr Ala Val Pro Thr Val Tyr Ty - #r His Pro Arg Ile Ser           345                 3 - #50                 3 - #55                 3 -      #60                                                                              - - CTG GCA TTT AAA GAT GGG TAT GCA ATA TGT AC - #T ATA GAA TGT GTT        CCC     3917                                                                    Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Th - #r Ile Glu Cys Val Pro                          365  - #               370  - #               375              - - TCT GGG ATT ACT GTG AGG TGG TTA GTT CAT GA - #T GAA CCC CAG CCT AAC         3965                                                                       Ser Gly Ile Thr Val Arg Trp Leu Val His As - #p Glu Pro Gln Pro Asn                       380      - #           385      - #           390                  - - ACA ACT TAT GAT ACT GTG GTT ACA GGT CTC TG - #C AGG ACC ATC GAT CGT         4013                                                                       Thr Thr Tyr Asp Thr Val Val Thr Gly Leu Cy - #s Arg Thr Ile Asp Arg                   395          - #       400          - #       405                      - - TAT AGA AAT CTC GCC AGT CGG ATT CCA GTC CA - #G GAC AAC TGG GCG AAA         4061                                                                       Tyr Arg Asn Leu Ala Ser Arg Ile Pro Val Gl - #n Asp Asn Trp Ala Lys               410              - #   415              - #   420                          - - ACG AAG TAT ACG TGC AGA CTA ATT GGA TAT CC - #G TTC GAC GTG GAT AGA         4109                                                                       Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pr - #o Phe Asp Val Asp Arg           425                 4 - #30                 4 - #35                 4 -      #40                                                                              - - TTT CAA AAT TCC GAA TAT TAT GAT GCA ACG CC - #G TCG GCA AGA GGA        ATG     4157                                                                    Phe Gln Asn Ser Glu Tyr Tyr Asp Ala Thr Pr - #o Ser Ala Arg Gly Met                          445  - #               450  - #               455              - - CCG ATG ATT GTA ACA ATT ACG GCC GTT CTA GG - #A CTG GCC TTG TTT TTA         4205                                                                       Pro Met Ile Val Thr Ile Thr Ala Val Leu Gl - #y Leu Ala Leu Phe Leu                       460      - #           465      - #           470                  - - GGT ATT GGT ATC ATT ATC ACA GCC CTA TGC TT - #T TAC CTA CCG GGG CGG         4253                                                                       Gly Ile Gly Ile Ile Ile Thr Ala Leu Cys Ph - #e Tyr Leu Pro Gly Arg                   475          - #       480          - #       485                      - - AAT TAAGATTAAC CATCGTATGT GATATAAAAA TTATTAAGTG TTATAACCG - #A              4306                                                                       Asn                                                                               490                                                                        - - TCGCATTCTT CTGTTTCGAT TCACAATAAA TAAAATGGTA TTGTAATCAG CA -             #CCATCGCA   4366                                                                 - - TTGTTTCGTA GATGACTCAT GTTCAGTCCG CGTGATGTCA AAAATACGTA TT -            #TTTGGTAT   4426                                                                 - - CACGCAGCGG CCAAAATGCC CATTATGTTA TTTTTACTCC AAACGCGGTA TT -            #TAAAACAT   4486                                                                 - - CGGGACGTAC ATCATGTGGC GCACGTTAAT CGTATACGGT GCCGCTACAT TA -            #AAAATCGC   4546                                                                 - - AAGTCTCCGA ATATCAAGCT CACGGCCAAA ACGTCGGTAA TAATCTTACG CA -            #TCGAATGT   4606                                                                 - - GATACGGATA CCGTACAATC GCTGAGTAGA TTTCCTATAT AGTTACTCAG TA -            #GTGATACA   4666                                                                 - - CAATCACAAA ATCGCTGGGG TATATCATAT AAGA ATG ATG TCG C - #CC ACC CCT           4718                                                                                        - #                  - #  Met Met Ser Pro Thr Pro                             - #                  - #    1              - # 5             - - GAA GAT GAT CGC GAT CTC GTT GTG GTT CGT GG - #A CGT CTC CGA ATG ATG         4766                                                                       Glu Asp Asp Arg Asp Leu Val Val Val Arg Gl - #y Arg Leu Arg Met Met                        10     - #             15     - #             20                  - - GAT AGC GGC ACG GAA ACA GAT AGA GAG CAA CG - #A CAT CCA CGT ACG ACT         4814                                                                       Asp Ser Gly Thr Glu Thr Asp Arg Glu Gln Ar - #g His Pro Arg Thr Thr                    25         - #         30         - #         35                      - - TGG CGA TCG ATC TGT TGT GGG TGT ACG ATA GG - #A ATG GTA TTT ACC ATA         4862                                                                       Trp Arg Ser Ile Cys Cys Gly Cys Thr Ile Gl - #y Met Val Phe Thr Ile                40             - #     45             - #     50                          - - TTC GTT CTC GTA GCG GCA GTA TTG TTG GGA TC - #A CTA TTC ACT GTT TCA         4910                                                                       Phe Val Leu Val Ala Ala Val Leu Leu Gly Se - #r Leu Phe Thr Val Ser            55                 - # 60                 - # 65                 - # 70       - - TAC ATG GCC ATG GAA TCG GGA ACA TGT CCC GA - #T GAA TGG ATT GGT TTG         4958                                                                       Tyr Met Ala Met Glu Ser Gly Thr Cys Pro As - #p Glu Trp Ile Gly Leu                            75 - #                 80 - #                 85              - - GGT TAT AGT TGC ATG CGC GTG GCC GGG AAA AA - #T GCA ACT GAT CTT GAG         5006                                                                       Gly Tyr Ser Cys Met Arg Val Ala Gly Lys As - #n Ala Thr Asp Leu Glu                        90     - #             95     - #            100                  - - GCG TTG GAT ACA TGT GCT CGG CAT AAC AGC AA - #A CTT ATT GAC TTC GCA         5054                                                                       Ala Leu Asp Thr Cys Ala Arg His Asn Ser Ly - #s Leu Ile Asp Phe Ala                   105          - #       110          - #       115                      - - AAC GCC AAA GTT CTG GTT GAA GCT ATC GCC CC - #A TTC GGT GTG CCA AAT         5102                                                                       Asn Ala Lys Val Leu Val Glu Ala Ile Ala Pr - #o Phe Gly Val Pro Asn               120              - #   125              - #   130                          - - GCA GCA TAT GGG GAA GTC TTC CGG TTA AGG GA - #C AGC AAA ACC ACG TGT         5150                                                                       Ala Ala Tyr Gly Glu Val Phe Arg Leu Arg As - #p Ser Lys Thr Thr Cys           135                 1 - #40                 1 - #45                 1 -      #50                                                                              - - ATA CGA CCT ACC ATG GGA GGA CCC GTG TCG GC - #A GAC TGT CCT GTA        ACA     5198                                                                    Ile Arg Pro Thr Met Gly Gly Pro Val Ser Al - #a Asp Cys Pro Val Thr                          155  - #               160  - #               165              - - TGT ACC GTT ATA TGT CAG CGA CCC AGG CCT CT - #A AGT ACC ATG TCT TCC         5246                                                                       Cys Thr Val Ile Cys Gln Arg Pro Arg Pro Le - #u Ser Thr Met Ser Ser                       170      - #           175      - #           180                  - - ATC ATT AGA GAT GCC CGC GTG TAT CTT CAT TT - #A GAA CGA CGC GAT TAT         5294                                                                       Ile Ile Arg Asp Ala Arg Val Tyr Leu His Le - #u Glu Arg Arg Asp Tyr                   185          - #       190          - #       195                      - - TAT GAA GTC TAC GCC TCT GTC CTC TCT AAT GC - #G ATG AGT AAA             TAAAAACGCA  5346                                                                Tyr Glu Val Tyr Ala Ser Val Leu Ser Asn Al - #a Met Ser Lys                       200              - #   205              - #   210                          - - CCTCTAACGG TTACTGTGTT TATTATCCAA TCACACCATA GACATTATTA CA -            #ATAATATG   5406                                                                 - - GATCTTTATT TCATATAATG            - #                  - #                     542 - #6                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 369 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:4:                     - - Met Met Ala Gly Ile Thr Val Ala Cys Asp Hi - #s Thr Ala Gly Glu Ala        1               5 - #                 10 - #                 15              - - His Thr Pro Glu Asp Met Gln Lys Lys Trp Ar - #g Ile Ile Leu Ala Gly                   20     - #             25     - #             30                  - - Glu Lys Phe Met Thr Ile Ser Ala Ser Leu Ly - #s Ser Ile Val Ser Cys               35         - #         40         - #         45                      - - Val Lys Asn Pro Leu Leu Thr Phe Gly Ala As - #p Gly Leu Ile Val Gln           50             - #     55             - #     60                          - - Gly Thr Val Cys Gly Gln Arg Ile Phe Val Pr - #o Ile Asp Arg Asp Ser       65                 - # 70                 - # 75                 - # 80       - - Phe Ser Glu Tyr Glu Trp His Gly Pro Thr Al - #a Met Phe Leu Ala Leu                       85 - #                 90 - #                 95              - - Thr Asp Ser Arg Arg Thr Leu Leu Asp Ala Ph - #e Lys Cys Glu Lys Arg                  100      - #           105      - #           110                  - - Arg Ala Ile Asp Val Ser Phe Thr Phe Ala Gl - #y Glu Pro Pro Cys Arg              115          - #       120          - #       125                      - - His Leu Ile Gln Ala Val Thr Tyr Met Thr As - #p Gly Gly Ser Val Ser          130              - #   135              - #   140                          - - Asn Thr Ile Ile Lys Tyr Glu Leu Trp Asn Al - #a Ser Thr Ile Phe Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gln Lys Thr Pro Asp Val Thr Phe Ser Leu As - #n Lys Gln Gln Leu        Asn                                                                                             165  - #               170  - #               175             - - Lys Ile Leu Ala Val Ala Ser Lys Leu Gln Hi - #s Glu Glu Leu Val Phe                  180      - #           185      - #           190                  - - Ser Leu Lys Pro Glu Gly Gly Phe Tyr Val Gl - #y Thr Val Cys Thr Val              195          - #       200          - #       205                      - - Ile Ser Phe Glu Val Asp Gly Thr Ala Met Th - #r Gln Tyr Pro Tyr Asn          210              - #   215              - #   220                          - - Pro Pro Thr Ser Ala Thr Leu Ala Leu Val Va - #l Ala Cys Arg Lys Lys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Lys Ala Asn Lys Asn Thr Ile Leu Thr Ala Ty - #r Gly Ser Gly Lys        Pro                                                                                             245  - #               250  - #               255             - - Phe Cys Val Ala Leu Glu Asp Thr Ser Ala Ph - #e Arg Asn Ile Val Asn                  260      - #           265      - #           270                  - - Lys Ile Lys Ala Gly Thr Ser Gly Val Asp Le - #u Gly Phe Tyr Thr Thr              275          - #       280          - #       285                      - - Cys Asp Pro Pro Met Leu Cys Ile Arg Pro Hi - #s Ala Phe Gly Ser Pro          290              - #   295              - #   300                          - - Thr Ala Phe Leu Phe Cys Asn Thr Asp Cys Me - #t Thr Ile Tyr Glu Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Glu Glu Val Ser Ala Val Asp Gly Ala Ile Ar - #g Ala Lys Arg Ile        Asn                                                                                             325  - #               330  - #               335             - - Glu Tyr Phe Pro Thr Val Ser Gln Ala Thr Se - #r Lys Lys Arg Lys Gln                  340      - #           345      - #           350                  - - Ser Pro Pro Pro Ile Glu Arg Glu Arg Lys Th - #r Thr Arg Ala Asp Thr              355          - #       360          - #       365                      - - Gln                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:5:                     - - Met Pro Ser Gly Ala Ser Ser Ser Pro Pro Pr - #o Ala Tyr Thr Ser Ala        1               5 - #                 10 - #                 15              - - Ala Pro Leu Glu Thr Tyr Asn Ser Trp Leu Se - #r Ala Phe Ser Cys Ala                   20     - #             25     - #             30                  - - Tyr Pro Gln Cys Thr Ala Gly Arg Gly His Ar - #g Gln Asn Gly Lys Lys               35         - #         40         - #         45                      - - Cys Ile Arg Cys Ile Val Ile Ser Val Cys Se - #r Leu Val Cys Ile Ala           50             - #     55             - #     60                          - - Ala His Leu Ala Val Thr Val Ser Gly Val Al - #a Leu Ile Pro Leu Ile       65                 - # 70                 - # 75                 - # 80       - - Asp Gln Asn Arg Ala Tyr Gly Asn Cys Thr Va - #l Cys Val Ile Ala Gly                       85 - #                 90 - #                 95              - - Phe Ile Ala Thr Phe Ala Ala Arg Leu Thr Il - #e Arg Leu Ser Glu Thr                  100      - #           105      - #           110                  - - Leu Met Leu Val Gly Lys Pro Ala Gln Phe Il - #e Phe Ala Ile Ile Ala              115          - #       120          - #       125                      - - Ser Val Ala Glu Thr Leu Ile Asn Asn Glu Al - #a Leu Ala Ile Ser Asn           130             - #    135             - #    140                         - - Thr Thr Tyr Lys Thr Ala Leu Arg Ile Ile Gl - #u Val Thr Ser Leu Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Phe Val Met Leu Gly Ala Ile Ile Thr Se - #r His Asn Tyr Val        Cys                                                                                             165  - #               170  - #               175             - - Ile Ser Thr Ala Gly Asp Leu Thr Trp Lys Gl - #y Gly Ile Phe His Ala                  180      - #           185      - #           190                  - - Tyr His Gly Thr Leu Leu Gly Ile Thr Ile Pr - #o Asn Ile His Pro Ile              195          - #       200          - #       205                      - - Pro Leu Ala Gly Phe Leu Ala Val Tyr Thr Il - #e Leu Ala Ile Asn Ile          210              - #   215              - #   220                          - - Ala Arg Asp Ala Ser Ala Thr Leu Leu Ser Th - #r Cys Tyr Tyr Arg Asn      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Cys Arg Glu Arg Thr Ile Leu Arg Pro Ser Ar - #g Leu Gly His Gly        Tyr                                                                                             245  - #               250  - #               255             - - Thr Ile Pro Ser Pro Gly Ala Asp Met Leu Ty - #r Glu Glu Asp Val Tyr                  260      - #           265      - #           270                  - - Ser Phe Asp Ala Ala Lys Gly His Tyr Ser Se - #r Ile Phe Leu Cys Tyr              275          - #       280          - #       285                      - - Ala Met Gly Leu Thr Thr Pro Leu Ile Ile Al - #a Leu His Lys Tyr Met          290              - #   295              - #   300                          - - Ala Gly Ile Lys Asn Ser Ser Asp Trp Thr Al - #a Thr Leu Gln Gly Met      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Tyr Gly Leu Val Leu Gly Ser Leu Ser Ser Le - #u Cys Ile Pro Ser        Ser                                                                                             325  - #               330  - #               335             - - Asn Asn Asp Ala Leu Ile Arg Pro Ile Gln Il - #e Leu Ile Leu Ile Ile                  340      - #           345      - #           350                  - - Gly Ala Leu Ala Ile Ala Leu Ala Gly Cys Gl - #y Gln Ile Ile Gly Pro              355          - #       360          - #       365                      - - Thr Leu Phe Ala Ala Ser Ser Ala Ala Met Se - #r Cys Phe Thr Cys Ile          370              - #   375              - #   380                          - - Asn Ile Arg Ala Thr Asn Lys Gly Val Asn Ly - #s Leu Ala Ala Ala Ser      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Val Val Lys Ser Val Leu Gly Phe Ile Ile Se - #r Gly Met Leu Thr        Cys                                                                                              405 - #                410 - #                415            - - Val Leu Leu Pro Leu Ser                                                              420                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:6:                     - - Met Val Ser Asn Met Arg Val Leu Arg Val Le - #u Arg Leu Thr Gly Trp        1               5 - #                 10 - #                 15              - - Val Gly Ile Phe Leu Val Leu Ser Leu Gln Gl - #n Thr Ser Cys Ala Gly                   20     - #             25     - #             30                  - - Leu Pro His Asn Val Asp Thr His His Ile Le - #u Thr Phe Asn Pro Ser               35         - #         40         - #         45                      - - Pro Ile Ser Ala Asp Gly Val Pro Leu Ser Gl - #u Val Pro Asn Ser Pro           50             - #     55             - #     60                          - - Thr Thr Glu Leu Ser Thr Thr Val Ala Thr Ly - #s Thr Ala Val Pro Thr       65                 - # 70                 - # 75                 - # 80       - - Thr Glu Ser Thr Ser Ser Ser Glu Ala His Ar - #g Asn Ser Ser His Lys                       85 - #                 90 - #                 95              - - Ile Pro Asp Ile Ile Cys Asp Arg Glu Glu Va - #l Phe Val Phe Leu Asn                  100      - #           105      - #           110                  - - Asn Thr Gly Arg Ile Leu Cys Asp Leu Ile Va - #l Asp Pro Pro Ser Asp              115          - #       120          - #       125                      - - Asp Glu Trp Ser Asn Phe Ala Leu Asp Val Th - #r Phe Asn Pro Ile Glu          130              - #   135              - #   140                          - - Tyr His Ala Asn Glu Lys Asn Val Glu Val Al - #a Arg Val Ala Gly Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Gly Val Pro Gly Ser Asp Tyr Ala Tyr Pr - #o Arg Lys Ser Glu        Leu                                                                                             165  - #               170  - #               175             - - Ile Ser Ser Ile Arg Arg Asp Pro Gln Gly Se - #r Phe Trp Thr Ser Pro                  180      - #           185      - #           190                  - - Thr Pro Arg Gly Asn Lys Tyr Phe Ile Trp Il - #e Asn Lys Thr Met His              195          - #       200          - #       205                      - - Thr Met Gly Val Glu Val Arg Asn Val Asp Ty - #r Lys Asp Asn Gly Tyr          210              - #   215              - #   220                          - - Phe Gln Val Ile Leu Arg Asp Arg Phe Asn Ar - #g Pro Leu Val Glu Lys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - His Ile Tyr Met Arg Val Cys Gln Arg Pro Al - #a Ser Val Asp Val        Leu                                                                                             245  - #               250  - #               255             - - Ala Pro Pro Val Leu Ser Gly Glu Asn Tyr Ly - #s Ala Ser Cys Ile Val                  260      - #           265      - #           270                  - - Arg His Phe Tyr Pro Pro Gly Ser Val Tyr Va - #l Ser Trp Arg Arg Asn              275          - #       280          - #       285                      - - Gly Asn Ile Ala Thr Pro Arg Lys Asp Arg As - #p Gly Ser Phe Trp Trp          290              - #   295              - #   300                          - - Phe Glu Ser Gly Arg Gly Ala Thr Leu Val Se - #r Thr Ile Thr Leu Gly      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asn Ser Gly Leu Glu Ser Pro Pro Lys Val Se - #r Cys Leu Val Ala        Trp                                                                                             325  - #               330  - #               335             - - Arg Gln Gly Asp Met Ile Ser Thr Ser Asn Al - #a Thr Ala Val Pro Thr                  340      - #           345      - #           350                  - - Val Tyr Tyr His Pro Arg Ile Ser Leu Ala Ph - #e Lys Asp Gly Tyr Ala              355          - #       360          - #       365                      - - Ile Cys Thr Ile Glu Cys Val Pro Ser Gly Il - #e Thr Val Arg Trp Leu          370              - #   375              - #   380                          - - Val His Asp Glu Pro Gln Pro Asn Thr Thr Ty - #r Asp Thr Val Val Thr      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gly Leu Cys Arg Thr Ile Asp Arg Tyr Arg As - #n Leu Ala Ser Arg        Ile                                                                                             405  - #               410  - #               415             - - Pro Val Gln Asp Asn Trp Ala Lys Thr Lys Ty - #r Thr Cys Arg Leu Ile                  420      - #           425      - #           430                  - - Gly Tyr Pro Phe Asp Val Asp Arg Phe Gln As - #n Ser Glu Tyr Tyr Asp              435          - #       440          - #       445                      - - Ala Thr Pro Ser Ala Arg Gly Met Pro Met Il - #e Val Thr Ile Thr Ala          450              - #   455              - #   460                          - - Val Leu Gly Leu Ala Leu Phe Leu Gly Ile Gl - #y Ile Ile Ile Thr Ala      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Leu Cys Phe Tyr Leu Pro Gly Arg Asn                                                      485                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 212 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:7:                     - - Met Met Ser Pro Thr Pro Glu Asp Asp Arg As - #p Leu Val Val Val        Arg                                                                               1               5 - #                 10 - #                 15             - - Gly Arg Leu Arg Met Met Asp Ser Gly Thr Gl - #u Thr Asp Arg Glu Gln                   20     - #             25     - #             30                  - - Arg His Pro Arg Thr Thr Trp Arg Ser Ile Cy - #s Cys Gly Cys Thr Ile               35         - #         40         - #         45                      - - Gly Met Val Phe Thr Ile Phe Val Leu Val Al - #a Ala Val Leu Leu Gly           50             - #     55             - #     60                          - - Ser Leu Phe Thr Val Ser Tyr Met Ala Met Gl - #u Ser Gly Thr Cys Pro       65                 - # 70                 - # 75                 - # 80       - - Asp Glu Trp Ile Gly Leu Gly Tyr Ser Cys Me - #t Arg Val Ala Gly Lys                       85 - #                 90 - #                 95              - - Asn Ala Thr Asp Leu Glu Ala Leu Asp Thr Cy - #s Ala Arg His Asn Ser                  100      - #           105      - #           110                  - - Lys Leu Ile Asp Phe Ala Asn Ala Lys Val Le - #u Val Glu Ala Ile Ala              115          - #       120          - #       125                      - - Pro Phe Gly Val Pro Asn Ala Ala Tyr Gly Gl - #u Val Phe Arg Leu Arg          130              - #   135              - #   140                          - - Asp Ser Lys Thr Thr Cys Ile Arg Pro Thr Me - #t Gly Gly Pro Val Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Asp Cys Pro Val Thr Cys Thr Val Ile Cy - #s Gln Arg Pro Arg        Pro                                                                                             165  - #               170  - #               175             - - Leu Ser Thr Met Ser Ser Ile Ile Arg Asp Al - #a Arg Val Tyr Leu His                  180      - #           185      - #           190                  - - Leu Glu Arg Arg Asp Tyr Tyr Glu Val Tyr Al - #a Ser Val Leu Ser Asn              195          - #       200          - #       205                      - - Ala Met Ser Lys                                                              210                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1506 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1506                                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:8:                        - - ATG CTC ACG CCG CGT GTG TTA CGA GCT TTG GG - #G TGG ACT GGA CTC TTT           48                                                                       Met Leu Thr Pro Arg Val Leu Arg Ala Leu Gl - #y Trp Thr Gly Leu Phe             1               5 - #                 10 - #                 15              - - TTT TTG CTT TTA TCT CCG AGC AAC GTC CTA GG - #A GCC AGC CTT AGC CGG           96                                                                       Phe Leu Leu Leu Ser Pro Ser Asn Val Leu Gl - #y Ala Ser Leu Ser Arg                        20     - #             25     - #             30                  - - GAT CTC GAA ACA CCC CCA TTT CTA TCC TTT GA - #T CCA TCC AAC ATT TCA          144                                                                       Asp Leu Glu Thr Pro Pro Phe Leu Ser Phe As - #p Pro Ser Asn Ile Ser                    35         - #         40         - #         45                      - - ATT AAC GGC GCG CCT TTA ACT GAG GTA CCT CA - #T GCA CCT TCC ACA GAA          192                                                                       Ile Asn Gly Ala Pro Leu Thr Glu Val Pro Hi - #s Ala Pro Ser Thr Glu                50             - #     55             - #     60                          - - AGT GTG TCA ACA AAT TCG GAA AGT ACC AAT GA - #A CAT ACC ATA ACA GAA          240                                                                       Ser Val Ser Thr Asn Ser Glu Ser Thr Asn Gl - #u His Thr Ile Thr Glu            65                 - # 70                 - # 75                 - # 80       - - ACG ACG GGC AAG AAC GCA TAC ATC CAC AAC AA - #T GCG TCT ACG GAC AAG          288                                                                       Thr Thr Gly Lys Asn Ala Tyr Ile His Asn As - #n Ala Ser Thr Asp Lys                            85 - #                 90 - #                 95              - - CAA AAT GCG AAC GAC ACT CAT AAA ACG CCC AA - #T ATA CTC TGC GAT ACG          336                                                                       Gln Asn Ala Asn Asp Thr His Lys Thr Pro As - #n Ile Leu Cys Asp Thr                       100      - #           105      - #           110                  - - GAA GAA GTT TTT GTT TTC CTT AAC GAA ACG GG - #A AGA TTT GTT TGT ACT          384                                                                       Glu Glu Val Phe Val Phe Leu Asn Glu Thr Gl - #y Arg Phe Val Cys Thr                   115          - #       120          - #       125                      - - CTC AAA GTC GAC CCC CCC TCG GAT AGT GAA TG - #G TCC AAC TTT GTT CTA          432                                                                       Leu Lys Val Asp Pro Pro Ser Asp Ser Glu Tr - #p Ser Asn Phe Val Leu               130              - #   135              - #   140                          - - GAT CTG ATC TTT AAC CCA ATT GAA TAC CAC GC - #C AAC GAA AAG AAT GTG          480                                                                       Asp Leu Ile Phe Asn Pro Ile Glu Tyr His Al - #a Asn Glu Lys Asn Val            145                 - #150                 - #155                 -         #160                                                                             - - GAA GCG GCG CGT ATC GCT GGT CTC TAT GGA GT - #C CCC GGA TCA GAC        TAT      528                                                                    Glu Ala Ala Arg Ile Ala Gly Leu Tyr Gly Va - #l Pro Gly Ser Asp Tyr                          165  - #               170  - #               175              - - GCA TAC CCA CGT CAA TCT GAA TTA ATT TCT TC - #G ATT CGA CGA GAT CCC          576                                                                       Ala Tyr Pro Arg Gln Ser Glu Leu Ile Ser Se - #r Ile Arg Arg Asp Pro                       180      - #           185      - #           190                  - - CAG GGC ACA TTT TGG ACG AGC CCA TCA CCT CA - #T GGA AAC AAG TAC TTC          624                                                                       Gln Gly Thr Phe Trp Thr Ser Pro Ser Pro Hi - #s Gly Asn Lys Tyr Phe                   195          - #       200          - #       205                      - - ATA TGG ATA AAC AAA ACA ACC AAT ACG ATG GG - #C GTG GAA ATT AGA AAT          672                                                                       Ile Trp Ile Asn Lys Thr Thr Asn Thr Met Gl - #y Val Glu Ile Arg Asn               210              - #   215              - #   220                          - - GTA GAT TAT GCT GAT AAT GGC TAC ATG CAA GT - #C ATT ATG CGT GAC CAT          720                                                                       Val Asp Tyr Ala Asp Asn Gly Tyr Met Gln Va - #l Ile Met Arg Asp His           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - TTT AAT CGG CCT TTA ATA GAT AAA CAT ATT TA - #C ATA CGT GTG TGT        CAA      768                                                                    Phe Asn Arg Pro Leu Ile Asp Lys His Ile Ty - #r Ile Arg Val Cys Gln                          245  - #               250  - #               255              - - CGA CCT GCA TCA GTG GAT GTA CTG GCC CCT CC - #A GTC CTC AGC GGA GAA          816                                                                       Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pr - #o Val Leu Ser Gly Glu                       260      - #           265      - #           270                  - - AAT TAC AAG GCA TCT TGT ATC GTT AGA CAC TT - #T TAT CCC CCT GGA TCT          864                                                                       Asn Tyr Lys Ala Ser Cys Ile Val Arg His Ph - #e Tyr Pro Pro Gly Ser                   275          - #       280          - #       285                      - - GTC TAT GTA TCT TGG AGA CAG AAT GGA AAC AT - #T GCA ACT CCT CGG AAA          912                                                                       Val Tyr Val Ser Trp Arg Gln Asn Gly Asn Il - #e Ala Thr Pro Arg Lys               290              - #   295              - #   300                          - - GAT CGC GAT GGA AGT TTT TGG TGG TTC GAA TC - #T GGT AGA GGA GCT ACG          960                                                                       Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Se - #r Gly Arg Gly Ala Thr           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - TTG GTT TCT ACA ATA ACA TTG GGA AAT TCA GG - #A ATT GAT TTC CCC        CCC     1008                                                                    Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gl - #y Ile Asp Phe Pro Pro                          325  - #               330  - #               335              - - AAA ATA TCT TGT CTG GTT GCC TGG AAG CAG GG - #T GAT ATG ATC AGC ACG         1056                                                                       Lys Ile Ser Cys Leu Val Ala Trp Lys Gln Gl - #y Asp Met Ile Ser Thr                       340      - #           345      - #           350                  - - ACG AAT GCC ACA GCT ATC CCG ACG GTA TAT CA - #T CAT CCC CGT TTA TCC         1104                                                                       Thr Asn Ala Thr Ala Ile Pro Thr Val Tyr Hi - #s His Pro Arg Leu Ser                   355          - #       360          - #       365                      - - CTG GCT TTT AAA GAT GGG TAT GCA ATA TGT AC - #T ATA GAA TGT GTC CCC         1152                                                                       Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Th - #r Ile Glu Cys Val Pro               370              - #   375              - #   380                          - - TCT GAG ATT ACT GTA CGG TGG TTA GTA CAT GA - #T GAA GCG CAG CCT AAC         1200                                                                       Ser Glu Ile Thr Val Arg Trp Leu Val His As - #p Glu Ala Gln Pro Asn           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - ACA ACT TAT AAT ACT GTG GTT ACA GGT CTC TG - #C CGG ACC ATC GAT        CGC     1248                                                                    Thr Thr Tyr Asn Thr Val Val Thr Gly Leu Cy - #s Arg Thr Ile Asp Arg                          405  - #               410  - #               415              - - CAT AGA AAT CTC CTC AGC CGC ATT CCA GTA TG - #G GAC AAT TGG ACG AAA         1296                                                                       His Arg Asn Leu Leu Ser Arg Ile Pro Val Tr - #p Asp Asn Trp Thr Lys                       420      - #           425      - #           430                  - - ACA AAA TAT ACG TGC AGA CTC ATA GGC TAC CC - #C TTC GAT GAA GAT AAA         1344                                                                       Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pr - #o Phe Asp Glu Asp Lys                   435          - #       440          - #       445                      - - TTT CAA GAT TCG GAA TAT TAC GAT GCA ACT CC - #A TCT GCA AGA GGA ACA         1392                                                                       Phe Gln Asp Ser Glu Tyr Tyr Asp Ala Thr Pr - #o Ser Ala Arg Gly Thr               450              - #   455              - #   460                          - - CCC ATG GTT ATT ACG GTT ACG GCA GTT TTG GG - #A TTG GCT GTA ATT TTA         1440                                                                       Pro Met Val Ile Thr Val Thr Ala Val Leu Gl - #y Leu Ala Val Ile Leu           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - GGG ATG GGG ATA ATC ATG ACT GCC CTA TGT TT - #A TAC AAC TCC ACA        CGA     1488                                                                    Gly Met Gly Ile Ile Met Thr Ala Leu Cys Le - #u Tyr Asn Ser Thr Arg                          485  - #               490  - #               495              - - AAA AAT ATT CGA TTA TAA         - #                  - #                      - #1506                                                                  Lys Asn Ile Arg Leu                                                                       500                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 501 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:9:                     - - Met Leu Thr Pro Arg Val Leu Arg Ala Leu Gl - #y Trp Thr Gly Leu Phe        1               5 - #                 10 - #                 15              - - Phe Leu Leu Leu Ser Pro Ser Asn Val Leu Gl - #y Ala Ser Leu Ser Arg                   20     - #             25     - #             30                  - - Asp Leu Glu Thr Pro Pro Phe Leu Ser Phe As - #p Pro Ser Asn Ile Ser               35         - #         40         - #         45                      - - Ile Asn Gly Ala Pro Leu Thr Glu Val Pro Hi - #s Ala Pro Ser Thr Glu           50             - #     55             - #     60                          - - Ser Val Ser Thr Asn Ser Glu Ser Thr Asn Gl - #u His Thr Ile Thr Glu       65                 - # 70                 - # 75                 - # 80       - - Thr Thr Gly Lys Asn Ala Tyr Ile His Asn As - #n Ala Ser Thr Asp Lys                       85 - #                 90 - #                 95              - - Gln Asn Ala Asn Asp Thr His Lys Thr Pro As - #n Ile Leu Cys Asp Thr                  100      - #           105      - #           110                  - - Glu Glu Val Phe Val Phe Leu Asn Glu Thr Gl - #y Arg Phe Val Cys Thr              115          - #       120          - #       125                      - - Leu Lys Val Asp Pro Pro Ser Asp Ser Glu Tr - #p Ser Asn Phe Val Leu          130              - #   135              - #   140                          - - Asp Leu Ile Phe Asn Pro Ile Glu Tyr His Al - #a Asn Glu Lys Asn Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Ala Ala Arg Ile Ala Gly Leu Tyr Gly Va - #l Pro Gly Ser Asp        Tyr                                                                                             165  - #               170  - #               175             - - Ala Tyr Pro Arg Gln Ser Glu Leu Ile Ser Se - #r Ile Arg Arg Asp Pro                  180      - #           185      - #           190                  - - Gln Gly Thr Phe Trp Thr Ser Pro Ser Pro Hi - #s Gly Asn Lys Tyr Phe              195          - #       200          - #       205                      - - Ile Trp Ile Asn Lys Thr Thr Asn Thr Met Gl - #y Val Glu Ile Arg Asn          210              - #   215              - #   220                          - - Val Asp Tyr Ala Asp Asn Gly Tyr Met Gln Va - #l Ile Met Arg Asp His      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Phe Asn Arg Pro Leu Ile Asp Lys His Ile Ty - #r Ile Arg Val Cys        Gln                                                                                             245  - #               250  - #               255             - - Arg Pro Ala Ser Val Asp Val Leu Ala Pro Pr - #o Val Leu Ser Gly Glu                  260      - #           265      - #           270                  - - Asn Tyr Lys Ala Ser Cys Ile Val Arg His Ph - #e Tyr Pro Pro Gly Ser              275          - #       280          - #       285                      - - Val Tyr Val Ser Trp Arg Gln Asn Gly Asn Il - #e Ala Thr Pro Arg Lys          290              - #   295              - #   300                          - - Asp Arg Asp Gly Ser Phe Trp Trp Phe Glu Se - #r Gly Arg Gly Ala Thr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Leu Val Ser Thr Ile Thr Leu Gly Asn Ser Gl - #y Ile Asp Phe Pro        Pro                                                                                             325  - #               330  - #               335             - - Lys Ile Ser Cys Leu Val Ala Trp Lys Gln Gl - #y Asp Met Ile Ser Thr                  340      - #           345      - #           350                  - - Thr Asn Ala Thr Ala Ile Pro Thr Val Tyr Hi - #s His Pro Arg Leu Ser              355          - #       360          - #       365                      - - Leu Ala Phe Lys Asp Gly Tyr Ala Ile Cys Th - #r Ile Glu Cys Val Pro          370              - #   375              - #   380                          - - Ser Glu Ile Thr Val Arg Trp Leu Val His As - #p Glu Ala Gln Pro Asn      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Thr Thr Tyr Asn Thr Val Val Thr Gly Leu Cy - #s Arg Thr Ile Asp        Arg                                                                                             405  - #               410  - #               415             - - His Arg Asn Leu Leu Ser Arg Ile Pro Val Tr - #p Asp Asn Trp Thr Lys                  420      - #           425      - #           430                  - - Thr Lys Tyr Thr Cys Arg Leu Ile Gly Tyr Pr - #o Phe Asp Glu Asp Lys              435          - #       440          - #       445                      - - Phe Gln Asp Ser Glu Tyr Tyr Asp Ala Thr Pr - #o Ser Ala Arg Gly Thr          450              - #   455              - #   460                          - - Pro Met Val Ile Thr Val Thr Ala Val Leu Gl - #y Leu Ala Val Ile Leu      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Gly Met Gly Ile Ile Met Thr Ala Leu Cys Le - #u Tyr Asn Ser Thr        Arg                                                                                             485  - #               490  - #               495             - - Lys Asn Ile Arg Leu                                                                  500                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1734 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1734                                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:10:                       - - ATG GAC CGC GCC GTT AGC CAA GTT GCG TTA GA - #G AAT GAT GAA AGA GAG           48                                                                       Met Asp Arg Ala Val Ser Gln Val Ala Leu Gl - #u Asn Asp Glu Arg Glu             1               5 - #                 10 - #                 15              - - GCA AAA AAT ACA TGG CGC TTG ATA TTC CGG AT - #T GCA ATC TTA TTC TTA           96                                                                       Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Il - #e Ala Ile Leu Phe Leu                        20     - #             25     - #             30                  - - ACA GTA GTG ACC TTG GCT ATA TCT GTA GCC TC - #C CTT TTA TAT AGC ATG          144                                                                       Thr Val Val Thr Leu Ala Ile Ser Val Ala Se - #r Leu Leu Tyr Ser Met                    35         - #         40         - #         45                      - - GGG GCT AGC ACA CCT AGC GAT CTT GTA GGC AT - #A CCG ACT AGG ATT TCC          192                                                                       Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Il - #e Pro Thr Arg Ile Ser                50             - #     55             - #     60                          - - AGG GCA GAA GAA AAG ATT ACA TCT ACA CTT GG - #T TCC AAT CAA GAT GTA          240                                                                       Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gl - #y Ser Asn Gln Asp Val            65                 - # 70                 - # 75                 - # 80       - - GTA GAT AGG ATA TAT AAG CAA GTG GCC CTT GA - #G TCT CCA TTG GCA TTG          288                                                                       Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Gl - #u Ser Pro Leu Ala Leu                            85 - #                 90 - #                 95              - - TTA AAT ACT GAG ACC ACA ATT ATG AAC GCA AT - #A ACA TCT CTC TCT TAT          336                                                                       Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Il - #e Thr Ser Leu Ser Tyr                       100      - #           105      - #           110                  - - CAG ATT AAT GGA GCT GCA AAC AAC AGC GGG TG - #G GGG GCA CCT ATT CAT          384                                                                       Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Tr - #p Gly Ala Pro Ile His                   115          - #       120          - #       125                      - - GAC CCA GAT TAT ATA GGG GGG ATA GGC AAA GA - #A CTC ATT GTA GAT GAT          432                                                                       Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Gl - #u Leu Ile Val Asp Asp               130              - #   135              - #   140                          - - GCT AGT GAT GTC ACA TCA TTC TAT CCC TCT GC - #A TTT CAA GAA CAT CTG          480                                                                       Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Al - #a Phe Gln Glu His Leu           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - AAT TTT ATC CCG GCG CCT ACT ACA GGA TCA GG - #T TGC ACT CGA ATA        CCC      528                                                                    Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gl - #y Cys Thr Arg Ile Pro                           165 - #                170 - #                175             - - TCA TTT GAC ATG AGT GCT ACC CAT TAC TGC TA - #C ACC CAT AAT GTA ATA          576                                                                       Ser Phe Asp Met Ser Ala Thr His Tyr Cys Ty - #r Thr His Asn Val Ile                       180      - #           185      - #           190                  - - TTG TCT GGA TGC AGA GAT CAC TCA CAC TCA CA - #T CAG TAT TTA GCA CTT          624                                                                       Leu Ser Gly Cys Arg Asp His Ser His Ser Hi - #s Gln Tyr Leu Ala Leu                   195          - #       200          - #       205                      - - GGT GTG CTC CGG ACA TCT GCA ACA GGG AGG GT - #A TTC TTT TCT ACT CTG          672                                                                       Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Va - #l Phe Phe Ser Thr Leu               210              - #   215              - #   220                          - - CGT TCC ATC AAC CTG GAC GAC ACC CAA AAT CG - #G AAG TCT TGC AGT GTG          720                                                                       Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Ar - #g Lys Ser Cys Ser Val           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - AGT GCA ACT CCC CTG GGT TGT GAT ATG CTG TG - #C TCG AAA GCC ACG        GAG      768                                                                    Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cy - #s Ser Lys Ala Thr Glu                          245  - #               250  - #               255              - - ACA GAG GAA GAA GAT TAT AAC TCA GCT GTC CC - #T ACG CGG ATG GTA CAT          816                                                                       Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pr - #o Thr Arg Met Val His                       260      - #           265      - #           270                  - - GGG AGG TTA GGG TTC GAC GGC CAA TAT CAC GA - #A AAG GAC CTA GAT GTC          864                                                                       Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Gl - #u Lys Asp Leu Asp Val                   275          - #       280          - #       285                      - - ACA ACA TTA TTC GGG GAC TGG GTG GCC AAC TA - #C CCA GGA GTA GGG GGT          912                                                                       Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Ty - #r Pro Gly Val Gly Gly               290              - #   295              - #   300                          - - GGA TCT TTT ATT GAC AGC CGC GTG TGG TTC TC - #A GTC TAC GGA GGG TTA          960                                                                       Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Se - #r Val Tyr Gly Gly Leu           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - AAA CCC AAT ACA CCC AGT GAC ACT GTA CAG GA - #A GGG AAA TAT GTG        ATA     1008                                                                    Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Gl - #u Gly Lys Tyr Val Ile                          325  - #               330  - #               335              - - TAC AAG CGA TAC AAT GAC ACA TGC CCA GAT GA - #G CAA GAC TAC CAG ATT         1056                                                                       Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Gl - #u Gln Asp Tyr Gln Ile                       340      - #           345      - #           350                  - - CGA ATG GCC AAG TCT TCG TAT AAG CCT GGA CG - #G TTT GGT GGG AAA CGC         1104                                                                       Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Ar - #g Phe Gly Gly Lys Arg                   355          - #       360          - #       365                      - - ATA CAG CAG GCT ATC TTA TCT ATC AAA GTG TC - #A ACA TCC TTA GGC GAA         1152                                                                       Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Se - #r Thr Ser Leu Gly Glu               370              - #   375              - #   380                          - - GAC CCG GTA CTG ACT GTA CCG CCC AAC ACA GT - #C ACA CTC ATG GGG GCC         1200                                                                       Asp Pro Val Leu Thr Val Pro Pro Asn Thr Va - #l Thr Leu Met Gly Ala           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - GAA GGC AGA ATT CTC ACA GTA GGG ACA TCC CA - #T TTC TTG TAT CAG        CGA     1248                                                                    Glu Gly Arg Ile Leu Thr Val Gly Thr Ser Hi - #s Phe Leu Tyr Gln Arg                          405  - #               410  - #               415              - - GGG TCA TCA TAC TTC TCT CCC GCG TTA TTA TA - #T CCT ATG ACA GTC AGC         1296                                                                       Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Ty - #r Pro Met Thr Val Ser                       420      - #           425      - #           430                  - - AAC AAA ACA GCC ACT CTT CAT AGT CCT TAT AC - #A TTC AAT GCC TTC ACT         1344                                                                       Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Th - #r Phe Asn Ala Phe Thr                   435          - #       440          - #       445                      - - CGG CCA GGT AGT ATC CCT TGC CAG GCT TCA GC - #A AGA TGC CCC AAC TCA         1392                                                                       Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Al - #a Arg Cys Pro Asn Ser               450              - #   455              - #   460                          - - TGT GTT ACT GGA GTC TAT ACA GAT CCA TAT CC - #C CTA ATC TTC TAT AGA         1440                                                                       Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pr - #o Leu Ile Phe Tyr Arg           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - AAC CAC ACC TTG CGA GGG GTA TTC GGG ACA AT - #G CTT GAT GGT GAA        CAA     1488                                                                    Asn His Thr Leu Arg Gly Val Phe Gly Thr Me - #t Leu Asp Gly Glu Gln                          485  - #               490  - #               495              - - GCA AGA CTT AAC CCT GCG TCT GCA GTA TTC GA - #T AGC ACA TCC CGC AGT         1536                                                                       Ala Arg Leu Asn Pro Ala Ser Ala Val Phe As - #p Ser Thr Ser Arg Ser                       500      - #           505      - #           510                  - - CGC ATA ACT CGA GTG AGT TCA AGC AGC ATC AA - #A GCA GCA TAC ACA ACA         1584                                                                       Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Ly - #s Ala Ala Tyr Thr Thr                   515          - #       520          - #       525                      - - TCA ACT TGT TTT AAA GTG GTC AAG ACC AAT AA - #G ACC TAT TGT CTC AGC         1632                                                                       Ser Thr Cys Phe Lys Val Val Lys Thr Asn Ly - #s Thr Tyr Cys Leu Ser               530              - #   535              - #   540                          - - ATT GCT GAA ATA TCT AAT ACT CTC TTC GGA GA - #A TTC AGA ATC GTC CCG         1680                                                                       Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Gl - #u Phe Arg Ile Val Pro           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - TTA CTA GTT GAG ATC CTC AAA GAT GAC GGG GT - #T AGA GAA GCC AGG        TCT     1728                                                                    Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Va - #l Arg Glu Ala Arg Ser                          565  - #               570  - #               575              - - GGC TAG                - #                  - #                  -      #         1734                                                                  Gly                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 577 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:11:                    - - Met Asp Arg Ala Val Ser Gln Val Ala Leu Gl - #u Asn Asp Glu Arg        Glu                                                                               1               5 - #                 10 - #                 15             - - Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Il - #e Ala Ile Leu Phe Leu                   20     - #             25     - #             30                  - - Thr Val Val Thr Leu Ala Ile Ser Val Ala Se - #r Leu Leu Tyr Ser Met               35         - #         40         - #         45                      - - Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Il - #e Pro Thr Arg Ile Ser           50             - #     55             - #     60                          - - Arg Ala Glu Glu Lys Ile Thr Ser Thr Leu Gl - #y Ser Asn Gln Asp Val       65                 - # 70                 - # 75                 - # 80       - - Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Gl - #u Ser Pro Leu Ala Leu                       85 - #                 90 - #                 95              - - Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Il - #e Thr Ser Leu Ser Tyr                  100      - #           105      - #           110                  - - Gln Ile Asn Gly Ala Ala Asn Asn Ser Gly Tr - #p Gly Ala Pro Ile His              115          - #       120          - #       125                      - - Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Gl - #u Leu Ile Val Asp Asp          130              - #   135              - #   140                          - - Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Al - #a Phe Gln Glu His Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gl - #y Cys Thr Arg Ile        Pro                                                                                             165  - #               170  - #               175             - - Ser Phe Asp Met Ser Ala Thr His Tyr Cys Ty - #r Thr His Asn Val Ile                  180      - #           185      - #           190                  - - Leu Ser Gly Cys Arg Asp His Ser His Ser Hi - #s Gln Tyr Leu Ala Leu              195          - #       200          - #       205                      - - Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Va - #l Phe Phe Ser Thr Leu          210              - #   215              - #   220                          - - Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Ar - #g Lys Ser Cys Ser Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cy - #s Ser Lys Ala Thr        Glu                                                                                             245  - #               250  - #               255             - - Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pr - #o Thr Arg Met Val His                  260      - #           265      - #           270                  - - Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Gl - #u Lys Asp Leu Asp Val              275          - #       280          - #       285                      - - Thr Thr Leu Phe Gly Asp Trp Val Ala Asn Ty - #r Pro Gly Val Gly Gly          290              - #   295              - #   300                          - - Gly Ser Phe Ile Asp Ser Arg Val Trp Phe Se - #r Val Tyr Gly Gly Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Pro Asn Thr Pro Ser Asp Thr Val Gln Gl - #u Gly Lys Tyr Val        Ile                                                                                             325  - #               330  - #               335             - - Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Gl - #u Gln Asp Tyr Gln Ile                  340      - #           345      - #           350                  - - Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Ar - #g Phe Gly Gly Lys Arg              355          - #       360          - #       365                      - - Ile Gln Gln Ala Ile Leu Ser Ile Lys Val Se - #r Thr Ser Leu Gly Glu          370              - #   375              - #   380                          - - Asp Pro Val Leu Thr Val Pro Pro Asn Thr Va - #l Thr Leu Met Gly Ala      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Glu Gly Arg Ile Leu Thr Val Gly Thr Ser Hi - #s Phe Leu Tyr Gln        Arg                                                                                             405  - #               410  - #               415             - - Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Ty - #r Pro Met Thr Val Ser                  420      - #           425      - #           430                  - - Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Th - #r Phe Asn Ala Phe Thr              435          - #       440          - #       445                      - - Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Al - #a Arg Cys Pro Asn Ser          450              - #   455              - #   460                          - - Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pr - #o Leu Ile Phe Tyr Arg      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Asn His Thr Leu Arg Gly Val Phe Gly Thr Me - #t Leu Asp Gly Glu        Gln                                                                                             485  - #               490  - #               495             - - Ala Arg Leu Asn Pro Ala Ser Ala Val Phe As - #p Ser Thr Ser Arg Ser                  500      - #           505      - #           510                  - - Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Ly - #s Ala Ala Tyr Thr Thr              515          - #       520          - #       525                      - - Ser Thr Cys Phe Lys Val Val Lys Thr Asn Ly - #s Thr Tyr Cys Leu Ser          530              - #   535              - #   540                          - - Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Gl - #u Phe Arg Ile Val Pro      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Leu Leu Val Glu Ile Leu Lys Asp Asp Gly Va - #l Arg Glu Ala Arg        Ser                                                                                             565  - #               570  - #               575             - - Gly                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1662 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1662                                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:12:                       - - ATG GGC TCC AGA CCT TCT ACC AAG AAC CCA GC - #A CCT ATG ATG CTG ACT           48                                                                       Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Al - #a Pro Met Met Leu Thr             1               5 - #                 10 - #                 15              - - ATC CGG GTC GCG CTG GTA CTG AGT TGC ATC TG - #T CCG GCA AAC TCC ATT           96                                                                       Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cy - #s Pro Ala Asn Ser Ile                        20     - #             25     - #             30                  - - GAT GGC AGG CCT CTT GCA GCT GCA GGA ATT GT - #G GTT ACA GGA GAC AAA          144                                                                       Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Va - #l Val Thr Gly Asp Lys                    35         - #         40         - #         45                      - - GCA GTC AAC ATA TAC ACC TCA TCC CAG ACA GG - #A TCA ATC ATA GTT AAG          192                                                                       Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gl - #y Ser Ile Ile Val Lys                50             - #     55             - #     60                          - - CTC CTC CCG AAT CTG CCA AAG GAT AAG GAG GC - #A TGT GCG AAA GCC CCC          240                                                                       Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Al - #a Cys Ala Lys Ala Pro            65                 - # 70                 - # 75                 - # 80       - - TTG GAT GCA TAC AAC AGG ACA TTG ACC ACT TT - #G CTC ACC CCC CTT GGT          288                                                                       Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Le - #u Leu Thr Pro Leu Gly                            85 - #                 90 - #                 95              - - GAC TCT ATC CGT AGG ATA CAA GAG TCT GTG AC - #T ACA TCT GGA GGG GGG          336                                                                       Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Th - #r Thr Ser Gly Gly Gly                       100      - #           105      - #           110                  - - AGA CAG GGG CGC CTT ATA GGC GCC ATT ATT GG - #C GGT GTG GCT CTT GGG          384                                                                       Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gl - #y Gly Val Ala Leu Gly                   115          - #       120          - #       125                      - - GTT GCA ACT GCC GCA CAA ATA ACA GCG GCC GC - #A GCT CTG ATA CAA GCC          432                                                                       Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Al - #a Ala Leu Ile Gln Ala               130              - #   135              - #   140                          - - AAA CAA AAT GCT GCC AAC ATC CTC CGA CTT AA - #A GAG AGC ATT GCC GCA          480                                                                       Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Ly - #s Glu Ser Ile Ala Ala           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - ACC AAT GAG GCT GTG CAT GAG GTC ACT GAC GG - #A TTA TCG CAA CTA        GCA      528                                                                    Thr Asn Glu Ala Val His Glu Val Thr Asp Gl - #y Leu Ser Gln Leu Ala                          165  - #               170  - #               175              - - GTG GCA GTT GGG AAG ATG CAG CAG TTC GTT AA - #T GAC CAA TTT AAT AAA          576                                                                       Val Ala Val Gly Lys Met Gln Gln Phe Val As - #n Asp Gln Phe Asn Lys                       180      - #           185      - #           190                  - - ACA GCT CAG GAA TTA GAC TGC ATC AAA ATT GC - #A CAG CAA GTT GGT GTA          624                                                                       Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Al - #a Gln Gln Val Gly Val                   195          - #       200          - #       205                      - - GAG CTC AAC CTG TAC CTA ACC GAA TCG ACT AC - #A GTA TTC GGA CCA CAA          672                                                                       Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Th - #r Val Phe Gly Pro Gln               210              - #   215              - #   220                          - - ATC ACT TCA CCT GCC TTA AAC AAG CTG ACT AT - #T CAG GCA CTT TAC AAT          720                                                                       Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Il - #e Gln Ala Leu Tyr Asn           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - CTA GCT GGT GGG AAT ATG GAT TAC TTA TTG AC - #T AAG TTA GGT ATA        GGG      768                                                                    Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Th - #r Lys Leu Gly Ile Gly                          245  - #               250  - #               255              - - AAC AAT CAA CTC AGC TCA TTA ATC GGT AGC GG - #C TTA ATC ACC GGT AAC          816                                                                       Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gl - #y Leu Ile Thr Gly Asn                       260      - #           265      - #           270                  - - CCT ATT CTA TAC GAC TCA CAG ACT CAA CTC TT - #G GGT ATA CAG GTA ACT          864                                                                       Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Le - #u Gly Ile Gln Val Thr                   275          - #       280          - #       285                      - - CTA CCT TCA GTC GGG AAC CTA AAT AAT ATG CG - #T GCC ACC TAC TTG GAA          912                                                                       Leu Pro Ser Val Gly Asn Leu Asn Asn Met Ar - #g Ala Thr Tyr Leu Glu               290              - #   295              - #   300                          - - ACC TTA TCC GTA AGC ACA ACC AGG GGA TTT GC - #C TCG GCA CTT GTC CCA          960                                                                       Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Al - #a Ser Ala Leu Val Pro           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - AAA GTG GTG ACA CGG GTC GGT TCT GTG ATA GA - #A GAA CTT GAC ACC        TCA     1008                                                                    Lys Val Val Thr Arg Val Gly Ser Val Ile Gl - #u Glu Leu Asp Thr Ser                          325  - #               330  - #               335              - - TAC TGT ATA GAA ACT GAC TTA GAT TTA TAT TG - #T ACA AGA ATA GTA ACG         1056                                                                       Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cy - #s Thr Arg Ile Val Thr                       340      - #           345      - #           350                  - - TTC CCT ATG TCC CCT GGT ATT TAC TCC TGC TT - #G AGC GGC AAT ACA TCG         1104                                                                       Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Le - #u Ser Gly Asn Thr Ser                   355          - #       360          - #       365                      - - GCC TGT ATG TAC TCA AAG ACC GAA GGC GCA CT - #T ACT ACA CCA TAT ATG         1152                                                                       Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Le - #u Thr Thr Pro Tyr Met               370              - #   375              - #   380                          - - ACT ATC AAA GGC TCA GTC ATC GCT AAC TGC AA - #G ATG ACA ACA TGT AGA         1200                                                                       Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Ly - #s Met Thr Thr Cys Arg           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - TGT GTA AAC CCC CCG GGT ATC ATA TCG CAA AA - #C TAT GGA GAA GCC        GTG     1248                                                                    Cys Val Asn Pro Pro Gly Ile Ile Ser Gln As - #n Tyr Gly Glu Ala Val                          405  - #               410  - #               415              - - TCT CTA ATA GAT AAA CAA TCA TGC AAT GTT TT - #A TCC TTA GGC GGG ATA         1296                                                                       Ser Leu Ile Asp Lys Gln Ser Cys Asn Val Le - #u Ser Leu Gly Gly Ile                        420     - #            425     - #            430                 - - ACT TTA AGG CTC AGT GGG GAA TTC GAT GTA AC - #T TAT CAG AAG AAT ATC         1344                                                                       Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Th - #r Tyr Gln Lys Asn Ile                   435          - #       440          - #       445                      - - TCA ATA CAA GAT TCT CAA GTA ATA ATA ACA GG - #C AAT CTT GAT ATC TCA         1392                                                                       Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gl - #y Asn Leu Asp Ile Ser               450              - #   455              - #   460                          - - ACT GAG CTT GGG AAT GTC AAC AAC TCG ATC AG - #T AAT GCC TTG AAT AAG         1440                                                                       Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Se - #r Asn Ala Leu Asn Lys           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - TTA GAG GAA AGC AAC AGA AAA CTA GAC AAA GT - #C AAT GTC AAA CTG        ACC     1488                                                                    Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Va - #l Asn Val Lys Leu Thr                          485  - #               490  - #               495              - - AGC ACA TCT GCT CTC ATT ACC TAT ATC GTT TT - #G ACT ATC ATA TCT CTT         1536                                                                       Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Le - #u Thr Ile Ile Ser Leu                       500      - #           505      - #           510                  - - GTT TTT GGT ATA CTT AGC CTG ATT CTA GCA TG - #C TAC CTA ATG TAC AAG         1584                                                                       Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cy - #s Tyr Leu Met Tyr Lys                   515          - #       520          - #       525                      - - CAA AAG GCG CAA CAA AAG ACC TTA TTA TGG CT - #T GGG AAT AAT ACC CTA         1632                                                                       Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Le - #u Gly Asn Asn Thr Leu               530              - #   535              - #   540                          - - GAT CAG ATG AGA GCC ACT ACA AAA ATG TGA  - #                  - #             1662                                                                     Asp Gln Met Arg Ala Thr Thr Lys Met                                           545                 5 - #50                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 553 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:13:                    - - Met Gly Ser Arg Pro Ser Thr Lys Asn Pro Al - #a Pro Met Met Leu Thr        1               5 - #                 10 - #                 15              - - Ile Arg Val Ala Leu Val Leu Ser Cys Ile Cy - #s Pro Ala Asn Ser Ile                   20     - #             25     - #             30                  - - Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Va - #l Val Thr Gly Asp Lys               35         - #         40         - #         45                      - - Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gl - #y Ser Ile Ile Val Lys           50             - #     55             - #     60                          - - Leu Leu Pro Asn Leu Pro Lys Asp Lys Glu Al - #a Cys Ala Lys Ala Pro       65                 - # 70                 - # 75                 - # 80       - - Leu Asp Ala Tyr Asn Arg Thr Leu Thr Thr Le - #u Leu Thr Pro Leu Gly                       85 - #                 90 - #                 95              - - Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Th - #r Thr Ser Gly Gly Gly                  100      - #           105      - #           110                  - - Arg Gln Gly Arg Leu Ile Gly Ala Ile Ile Gl - #y Gly Val Ala Leu Gly              115          - #       120          - #       125                      - - Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Al - #a Ala Leu Ile Gln Ala          130              - #   135              - #   140                          - - Lys Gln Asn Ala Ala Asn Ile Leu Arg Leu Ly - #s Glu Ser Ile Ala Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Asn Glu Ala Val His Glu Val Thr Asp Gl - #y Leu Ser Gln Leu        Ala                                                                                             165  - #               170  - #               175             - - Val Ala Val Gly Lys Met Gln Gln Phe Val As - #n Asp Gln Phe Asn Lys                   180     - #            185     - #            190                 - - Thr Ala Gln Glu Leu Asp Cys Ile Lys Ile Al - #a Gln Gln Val Gly Val              195          - #       200          - #       205                      - - Glu Leu Asn Leu Tyr Leu Thr Glu Ser Thr Th - #r Val Phe Gly Pro Gln          210              - #   215              - #   220                          - - Ile Thr Ser Pro Ala Leu Asn Lys Leu Thr Il - #e Gln Ala Leu Tyr Asn      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Th - #r Lys Leu Gly Ile        Gly                                                                                             245  - #               250  - #               255             - - Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gl - #y Leu Ile Thr Gly Asn                  260      - #           265      - #           270                  - - Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Le - #u Gly Ile Gln Val Thr              275          - #       280          - #       285                      - - Leu Pro Ser Val Gly Asn Leu Asn Asn Met Ar - #g Ala Thr Tyr Leu Glu          290              - #   295              - #   300                          - - Thr Leu Ser Val Ser Thr Thr Arg Gly Phe Al - #a Ser Ala Leu Val Pro      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Val Val Thr Arg Val Gly Ser Val Ile Gl - #u Glu Leu Asp Thr        Ser                                                                                             325  - #               330  - #               335             - - Tyr Cys Ile Glu Thr Asp Leu Asp Leu Tyr Cy - #s Thr Arg Ile Val Thr                  340      - #           345      - #           350                  - - Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Le - #u Ser Gly Asn Thr Ser              355          - #       360          - #       365                      - - Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Le - #u Thr Thr Pro Tyr Met          370              - #   375              - #   380                          - - Thr Ile Lys Gly Ser Val Ile Ala Asn Cys Ly - #s Met Thr Thr Cys Arg      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Cys Val Asn Pro Pro Gly Ile Ile Ser Gln As - #n Tyr Gly Glu Ala        Val                                                                                             405  - #               410  - #               415             - - Ser Leu Ile Asp Lys Gln Ser Cys Asn Val Le - #u Ser Leu Gly Gly Ile                  420      - #           425      - #           430                  - - Thr Leu Arg Leu Ser Gly Glu Phe Asp Val Th - #r Tyr Gln Lys Asn Ile              435          - #       440          - #       445                      - - Ser Ile Gln Asp Ser Gln Val Ile Ile Thr Gl - #y Asn Leu Asp Ile Ser           450             - #    455             - #    460                         - - Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Se - #r Asn Ala Leu Asn Lys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Leu Glu Glu Ser Asn Arg Lys Leu Asp Lys Va - #l Asn Val Lys Leu        Thr                                                                                             485  - #               490  - #               495             - - Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Le - #u Thr Ile Ile Ser Leu                  500      - #           505      - #           510                  - - Val Phe Gly Ile Leu Ser Leu Ile Leu Ala Cy - #s Tyr Leu Met Tyr Lys              515          - #       520          - #       525                      - - Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Le - #u Gly Asn Asn Thr Leu          530              - #   535              - #   540                          - - Asp Gln Met Arg Ala Thr Thr Lys Met                                      545                 5 - #50                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3489 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3489                                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:14:                       - - ATG TTG GTA ACA CCT CTT TTA CTA GTG ACT CT - #T TTG TGT GTA CTA TGT           48                                                                       Met Leu Val Thr Pro Leu Leu Leu Val Thr Le - #u Leu Cys Val Leu Cys             1               5 - #                 10 - #                 15              - - AGT GCT GCT TTG TAT GAC AGT AGT TCT TAC GT - #T TAC TAC TAC CAA AGT           96                                                                       Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Va - #l Tyr Tyr Tyr Gln Ser                        20     - #             25     - #             30                  - - GCC TTT AGA CCA CCT AAT GGT TGG CAT TTA CA - #C GGG GGT GCT TAT GCG          144                                                                       Ala Phe Arg Pro Pro Asn Gly Trp His Leu Hi - #s Gly Gly Ala Tyr Ala                    35         - #         40         - #         45                      - - GTA GTT AAT ATT TCT AGC GAA TCT AAT AAT GC - #A GGC TCT TCA CCT GGG          192                                                                       Val Val Asn Ile Ser Ser Glu Ser Asn Asn Al - #a Gly Ser Ser Pro Gly                50             - #     55             - #     60                          - - TGT ATT GTT GGT ACT ATT CAT GGT GGT CGT GT - #T GTT AAT GCT TCT TCT          240                                                                       Cys Ile Val Gly Thr Ile His Gly Gly Arg Va - #l Val Asn Ala Ser Ser            65                 - # 70                 - # 75                 - # 80       - - ATA GCT ATG ACG GCA CCG TCA TCA GGT ATG GC - #T TGG TCT AGC AGT CAG          288                                                                       Ile Ala Met Thr Ala Pro Ser Ser Gly Met Al - #a Trp Ser Ser Ser Gln                            85 - #                 90 - #                 95              - - TTT TGT ACT GCA CAC TGT AAC TTT TCA GAT AC - #T ACA GTG TTT GTT ACA          336                                                                       Phe Cys Thr Ala His Cys Asn Phe Ser Asp Th - #r Thr Val Phe Val Thr                       100      - #           105      - #           110                  - - CAT TGT TAT AAA TAT GAT GGG TGT CCT ATA AC - #T GGC ATG CTT CAA AAG          384                                                                       His Cys Tyr Lys Tyr Asp Gly Cys Pro Ile Th - #r Gly Met Leu Gln Lys                   115          - #       120          - #       125                      - - AAT TTT TTA CGT GTT TCT GCT ATG AAA AAT GG - #C CAG CTT TTC TAT AAT          432                                                                       Asn Phe Leu Arg Val Ser Ala Met Lys Asn Gl - #y Gln Leu Phe Tyr Asn                130             - #    135             - #    140                         - - TTA ACA GTT AGT GTA GCT AAG TAC CCT ACT TT - #T AAA TCA TTT CAG TGT          480                                                                       Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Ph - #e Lys Ser Phe Gln Cys           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GTT AAT AAT TTA ACA TCC GTA TAT TTA AAT GG - #T GAT CTT GTT TAC        ACC      528                                                                    Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gl - #y Asp Leu Val Tyr Thr                          165  - #               170  - #               175              - - TCT AAT GAG ACC ACA GAT GTT ACA TCT GCA GG - #T GTT TAT TTT AAA GCT          576                                                                       Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gl - #y Val Tyr Phe Lys Ala                       180      - #           185      - #           190                  - - GGT GGA CCT ATA ACT TAT AAA GTT ATG AGA AA - #A GTT AAA GCC CTG GCT          624                                                                       Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Ly - #s Val Lys Ala Leu Ala                   195          - #       200          - #       205                      - - TAT TTT GTT AAT GGT ACT GCA CAA GAT GTT AT - #T TTG TGT GAT GGA TCA          672                                                                       Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Il - #e Leu Cys Asp Gly Ser               210              - #   215              - #   220                          - - CCT AGA GGC TTG TTA GCA TGC CAG TAT AAT AC - #T GGC AAT TTT TCA GAT          720                                                                       Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Th - #r Gly Asn Phe Ser Asp           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GGC TTT TAT CCT TTT ATT AAT AGT AGT TTA GT - #T AAG CAG AAG TTT        ATT      768                                                                    Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Va - #l Lys Gln Lys Phe Ile                          245  - #               250  - #               255              - - GTC TAT CGT GAA AAT AGT GTT AAT ACT ACT TT - #T ACG TTA CAC AAT TTC          816                                                                       Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Ph - #e Thr Leu His Asn Phe                       260      - #           265      - #           270                  - - ACT TTT CAT AAT GAG ACT GGC GCC AAC CCT AA - #T CCT AGT GGT GTT CAG          864                                                                       Thr Phe His Asn Glu Thr Gly Ala Asn Pro As - #n Pro Ser Gly Val Gln                   275          - #       280          - #       285                      - - AAT ATT CTA ACT TAC CAA ACA CAA ACA GCT CA - #G AGT GGT TAT TAT AAT          912                                                                       Asn Ile Leu Thr Tyr Gln Thr Gln Thr Ala Gl - #n Ser Gly Tyr Tyr Asn               290              - #   295              - #   300                          - - TTT AAT TTT TCC TTT CTG AGT AGT TTT GTT TA - #T AAG GAG TCT AAT TTT          960                                                                       Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Ty - #r Lys Glu Ser Asn Phe           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ATG TAT GGA TCT TAT CAC CCA AGT TGT AAT TT - #T AGA CTA GAA ACT        ATT     1008                                                                    Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Ph - #e Arg Leu Glu Thr Ile                          325  - #               330  - #               335              - - AAT AAT GGC TTG TGG TTT AAT TCA CTT TCA GT - #T TCA ATT GCT TAC GGT         1056                                                                       Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Va - #l Ser Ile Ala Tyr Gly                       340      - #           345      - #           350                  - - CCT CTT CAA GGT GGT TGC AAG CAA TCT GTC TT - #T AGT GGT AGA GCA ACT         1104                                                                       Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Ph - #e Ser Gly Arg Ala Thr                   355          - #       360          - #       365                      - - TGT TGT TAT GCT TAT TCA TAT GGA GGT CCT TC - #G CTG TGT AAA GGT GTT         1152                                                                       Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Se - #r Leu Cys Lys Gly Val               370              - #   375              - #   380                          - - TAT TCA GGT GAG TTA GAT CTT AAT TTT GAA TG - #T GGA CTG TTA GTT TAT         1200                                                                       Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cy - #s Gly Leu Leu Val Tyr           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - GTT ACT AAG AGC GGT GGC TCT CGT ATA CAA AC - #A GCC ACT GAA CCG        CCA     1248                                                                    Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Th - #r Ala Thr Glu Pro Pro                          405  - #               410  - #               415              - - GTT ATA ACT CGA CAC AAT TAT AAT AAT ATT AC - #T TTA AAT ACT TGT GTT         1296                                                                       Val Ile Thr Arg His Asn Tyr Asn Asn Ile Th - #r Leu Asn Thr Cys Val                       420      - #           425      - #           430                  - - GAT TAT AAT ATA TAT GGC AGA ACT GGC CAA GG - #T TTT ATT ACT AAT GTA         1344                                                                       Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gl - #y Phe Ile Thr Asn Val                   435          - #       440          - #       445                      - - ACC GAC TCA GCT GTT AGT TAT AAT TAT CTA GC - #A GAC GCA GGT TTG GCT         1392                                                                       Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Al - #a Asp Ala Gly Leu Ala               450              - #   455              - #   460                          - - ATT TTA GAT ACA TCT GGT TCC ATA GAC ATC TT - #T GTT GTA CAA GGT GAA         1440                                                                       Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Ph - #e Val Val Gln Gly Glu           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - TAT GGT CTT ACT TAT TAT AAG GTT AAC CCT TG - #C GAA GAT GTC AAC        CAG     1488                                                                    Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cy - #s Glu Asp Val Asn Gln                          485  - #               490  - #               495              - - CAG TTT GTA GTT TCT GGT GGT AAA TTA GTA GG - #T ATT CTT ACT TCA CGT         1536                                                                       Gln Phe Val Val Ser Gly Gly Lys Leu Val Gl - #y Ile Leu Thr Ser Arg                       500      - #           505      - #           510                  - - AAT GAG ACT GGT TCT CAG CTT CTT GAG AAC CA - #G TTT TAC ATT AAA ATC         1584                                                                       Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gl - #n Phe Tyr Ile Lys Ile                   515          - #       520          - #       525                      - - ACT AAT GGA ACA CGT CGT TTT AGA CGT TCT AT - #T ACT GAA AAT GTT GCA         1632                                                                       Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Il - #e Thr Glu Asn Val Ala               530              - #   535              - #   540                          - - AAT TGC CCT TAT GTT AGT TAT GGT AAG TTT TG - #T ATA AAA CCT GAT GGT         1680                                                                       Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cy - #s Ile Lys Pro Asp Gly           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - TCA ATT GCC ACA ATA GTA CCA AAA CAA TTG GA - #A CAG TTT GTG GCA        CCT     1728                                                                    Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Gl - #u Gln Phe Val Ala Pro                          565  - #               570  - #               575              - - TTA CTT AAT GTT ACT GAA AAT GTG CTC ATA CC - #T AAC AGT TTT AAT TTA         1776                                                                       Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pr - #o Asn Ser Phe Asn Leu                       580      - #           585      - #           590                  - - ACT GTT ACA GAT GAG TAC ATA CAA ACG CGT AT - #G GAT AAG GTC CAA ATT         1824                                                                       Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Me - #t Asp Lys Val Gln Ile                   595          - #       600          - #       605                      - - AAT TGT CTG CAG TAT GTT TGT GGC AAT TCT CT - #G GAT TGT AGA GAT TTG         1872                                                                       Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Le - #u Asp Cys Arg Asp Leu               610              - #   615              - #   620                          - - TTT CAA CAA TAT GGG CCT GTT TGT GAC AAC AT - #A TTG TCT GTA GTA AAT         1920                                                                       Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Il - #e Leu Ser Val Val Asn           625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - AGT ATT GGT CAA AAA GAA GAT ATG GAA CTT TT - #G AAT TTC TAT TCT        TCT     1968                                                                    Ser Ile Gly Gln Lys Glu Asp Met Glu Leu Le - #u Asn Phe Tyr Ser Ser                          645  - #               650  - #               655              - - ACT AAA CCG GCT GGT TTT AAT ACA CCA TTT CT - #T AGT AAT GTT AGC ACT         2016                                                                       Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Le - #u Ser Asn Val Ser Thr                       660      - #           665      - #           670                  - - GGT GAG TTT AAT ATT TCT CTT CTG TTA ACA AC - #T CCT AGT AGT CCT AGA         2064                                                                       Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Th - #r Pro Ser Ser Pro Arg                   675          - #       680          - #       685                      - - AGG CGT TCT TTT ATT GAA GAC CTT CTA TTT AC - #A AGC GTT GAA TCT GTT         2112                                                                       Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Th - #r Ser Val Glu Ser Val               690              - #   695              - #   700                          - - GGA TTA CCA ACA GAT GAC GCA TAC AAA AAT TG - #C ACT GCA GGA CCT TTA         2160                                                                       Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cy - #s Thr Ala Gly Pro Leu           705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - GGT TTT CTT AAG GAC CTT GCG TGT GCT CGT GA - #A TAT AAT GGT TTG        CTT     2208                                                                    Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Gl - #u Tyr Asn Gly Leu Leu                           725 - #                730 - #                735             - - GTG TTG CCT CCC ATT ATA ACA GCA GAA ATG CA - #A ACT TTG TAT ACT AGT         2256                                                                       Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gl - #n Thr Leu Tyr Thr Ser                       740      - #           745      - #           750                  - - TCT CTA GTA GCT TCT ATG GCT TTT GGT GGT AT - #T ACT GCA GCT GGT GCT         2304                                                                       Ser Leu Val Ala Ser Met Ala Phe Gly Gly Il - #e Thr Ala Ala Gly Ala                   755          - #       760          - #       765                      - - ATA CCT TTT GCC ACA CAA CTG CAG GCT AGA AT - #T AAT CAC TTG GGT ATT         2352                                                                       Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Il - #e Asn His Leu Gly Ile               770              - #   775              - #   780                          - - ACC CAG TCA CTT TTG TTG AAG AAT CAA GAA AA - #A ATT GCT GCT TCC TTT         2400                                                                       Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Ly - #s Ile Ala Ala Ser Phe           785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - AAT AAG GCC ATT GGT CGT ATG CAG GAA GGT TT - #T AGA AGT ACA TCT        CTA     2448                                                                    Asn Lys Ala Ile Gly Arg Met Gln Glu Gly Ph - #e Arg Ser Thr Ser Leu                          805  - #               810  - #               815              - - GCA TTA CAA CAA ATT CAA GAT GTT GTT AAT AA - #G CAG AGT GCT ATT CTT         2496                                                                       Ala Leu Gln Gln Ile Gln Asp Val Val Asn Ly - #s Gln Ser Ala Ile Leu                       820      - #           825      - #           830                  - - ACT GAG ACT ATG GCA TCA CTT AAT AAA AAT TT - #T GGT GCT ATT TCT TCT         2544                                                                       Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Ph - #e Gly Ala Ile Ser Ser                   835          - #       840          - #       845                      - - GTG ATT CAA GAA ATC TAC CAG CAA CTT GAC GC - #C ATA CAA GCA AAT GCT         2592                                                                       Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Al - #a Ile Gln Ala Asn Ala               850              - #   855              - #   860                          - - CAA GTG GAT CGT CTT ATA ACT GGT AGA TTG TC - #A TCA CTT TCT GTT TTA         2640                                                                       Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Se - #r Ser Leu Ser Val Leu           865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - GCA TCT GCT AAG CAG GCG GAG CAT ATT AGA GT - #G TCA CAA CAG CGT        GAG     2688                                                                    Ala Ser Ala Lys Gln Ala Glu His Ile Arg Va - #l Ser Gln Gln Arg Glu                          885  - #               890  - #               895              - - TTA GCT ACT CAG AAA ATT AAT GAG TGT GTT AA - #G TCA CAG TCT ATT AGG         2736                                                                       Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Ly - #s Ser Gln Ser Ile Arg                       900      - #           905      - #           910                  - - TAC TCC TTT TGT GGT AAT GGA CGA CAT GTT CT - #A ACC ATA CCG CAA AAT         2784                                                                       Tyr Ser Phe Cys Gly Asn Gly Arg His Val Le - #u Thr Ile Pro Gln Asn                   915          - #       920          - #       925                      - - GCA CCT AAT GGT ATA GTG TTT ATA CAC TTT TC - #T TAT ACT CCA GAT AGT         2832                                                                       Ala Pro Asn Gly Ile Val Phe Ile His Phe Se - #r Tyr Thr Pro Asp Ser               930              - #   935              - #   940                          - - TTT GTT AAT GTT ACT GCA ATA GTG GGT TTT TG - #T GTA AAG CCA GCT AAT         2880                                                                       Phe Val Asn Val Thr Ala Ile Val Gly Phe Cy - #s Val Lys Pro Ala Asn           945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - GCT AGT CAG TAT GCA ATA GTA CCC GCT AAT GG - #T AGG GGT ATT TTT        ATA     2928                                                                    Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gl - #y Arg Gly Ile Phe Ile                          965  - #               970  - #               975              - - CAA GTT AAT GGT AGT TAC TAC ATC ACA GCA CG - #A GAT ATG TAT ATG CCA         2976                                                                       Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Ar - #g Asp Met Tyr Met Pro                       980      - #           985      - #           990                  - - AGA GCT ATT ACT GCA GGA GAT ATA GTT ACG CT - #T ACT TCT TGT CAA GCA         3024                                                                       Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Le - #u Thr Ser Cys Gln Ala                   995          - #       1000          - #      1005                     - - AAT TAT GTA AGT GTA AAT AAG ACC GTC ATT AC - #T ACA TTC GTA GAC AAT         3072                                                                       Asn Tyr Val Ser Val Asn Lys Thr Val Ile Th - #r Thr Phe Val Asp Asn               1010             - #   1015              - #  1020                         - - GAT GAT TTT GAT TTT AAT GAC GAA TTG TCA AA - #A TGG TGG AAT GAC ACT         3120                                                                       Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Ly - #s Trp Trp Asn Asp Thr           1025                1030 - #                1035 - #               1040        - - AAG CAT GAG CTA CCA GAC TTT GAC AAA TTC AA - #T TAC ACA GTA CCT ATA         3168                                                                       Lys His Glu Leu Pro Asp Phe Asp Lys Phe As - #n Tyr Thr Val Pro Ile                           1045 - #               1050  - #              1055             - - CTT GAC ATT GAT AGT GAA ATT GAT CGT ATT CA - #A GGC GTT ATA CAG GGT         3216                                                                       Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gl - #n Gly Val Ile Gln Gly                       1060     - #           1065      - #          1070                 - - CTT AAT GAC TCT TTA ATA GAC CTT GAA AAA CT - #T TCA ATA CTC AAA ACT         3264                                                                       Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Le - #u Ser Ile Leu Lys Thr                    1075        - #        1080         - #       1085                    - - TAT ATT AAG TGG CCT TGG TAT GTG TGG TTA GC - #C ATA GCT TTT GCC ACT         3312                                                                       Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Al - #a Ile Ala Phe Ala Thr               1090             - #   1095              - #  1100                         - - ATT ATC TTC ATC TTA ATA CTA GGA TGG GTT TT - #C TTC ATG ACT GGA TGT         3360                                                                       Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Ph - #e Phe Met Thr Gly Cys           1105                1110 - #                1115 - #               1120        - - TGT GGT TGT TGT TGT GGA TGC TTT GGC ATT AT - #G CCT CTA ATG AGT AAG         3408                                                                       Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Me - #t Pro Leu Met Ser Lys                           1125 - #               1130  - #              1135             - - TGT GGT AAG AAA TCT TCT TAT TAC ACG ACT TT - #T GAT AAC GAT GTG GTA         3456                                                                       Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Ph - #e Asp Asn Asp Val Val                       1140     - #           1145      - #          1150                 - - ACT GAA CAA AAC AGA CCT AAA AAG TCT GTT TA - #A                  -      #       3489                                                                    Thr Glu Gln Asn Arg Pro Lys Lys Ser Val                                               1155         - #       1160                                            - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1162 amino - #acids                                               (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:15:                    - - Met Leu Val Thr Pro Leu Leu Leu Val Thr Le - #u Leu Cys Val Leu        Cys                                                                               1               5 - #                 10 - #                 15             - - Ser Ala Ala Leu Tyr Asp Ser Ser Ser Tyr Va - #l Tyr Tyr Tyr Gln Ser                    20    - #              25    - #              30                 - - Ala Phe Arg Pro Pro Asn Gly Trp His Leu Hi - #s Gly Gly Ala Tyr Ala               35         - #         40         - #         45                      - - Val Val Asn Ile Ser Ser Glu Ser Asn Asn Al - #a Gly Ser Ser Pro Gly           50             - #     55             - #     60                          - - Cys Ile Val Gly Thr Ile His Gly Gly Arg Va - #l Val Asn Ala Ser Ser       65                 - # 70                 - # 75                 - # 80       - - Ile Ala Met Thr Ala Pro Ser Ser Gly Met Al - #a Trp Ser Ser Ser Gln                       85 - #                 90 - #                 95              - - Phe Cys Thr Ala His Cys Asn Phe Ser Asp Th - #r Thr Val Phe Val Thr                  100      - #           105      - #           110                  - - His Cys Tyr Lys Tyr Asp Gly Cys Pro Ile Th - #r Gly Met Leu Gln Lys              115          - #       120          - #       125                      - - Asn Phe Leu Arg Val Ser Ala Met Lys Asn Gl - #y Gln Leu Phe Tyr Asn          130              - #   135              - #   140                          - - Leu Thr Val Ser Val Ala Lys Tyr Pro Thr Ph - #e Lys Ser Phe Gln Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gl - #y Asp Leu Val Tyr        Thr                                                                                             165  - #               170  - #               175             - - Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gl - #y Val Tyr Phe Lys Ala                  180      - #           185      - #           190                  - - Gly Gly Pro Ile Thr Tyr Lys Val Met Arg Ly - #s Val Lys Ala Leu Ala              195          - #       200          - #       205                      - - Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Il - #e Leu Cys Asp Gly Ser          210              - #   215              - #   220                          - - Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Th - #r Gly Asn Phe Ser Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Va - #l Lys Gln Lys Phe        Ile                                                                                             245  - #               250  - #               255             - - Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Ph - #e Thr Leu His Asn Phe                  260      - #           265      - #           270                  - - Thr Phe His Asn Glu Thr Gly Ala Asn Pro As - #n Pro Ser Gly Val Gln              275          - #       280          - #       285                      - - Asn Ile Leu Thr Tyr Gln Thr Gln Thr Ala Gl - #n Ser Gly Tyr Tyr Asn           290             - #    295             - #    300                         - - Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Ty - #r Lys Glu Ser Asn Phe      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Ph - #e Arg Leu Glu Thr        Ile                                                                                             325  - #               330  - #               335             - - Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Va - #l Ser Ile Ala Tyr Gly                  340      - #           345      - #           350                  - - Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Ph - #e Ser Gly Arg Ala Thr              355          - #       360          - #       365                      - - Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Se - #r Leu Cys Lys Gly Val          370              - #   375              - #   380                          - - Tyr Ser Gly Glu Leu Asp Leu Asn Phe Glu Cy - #s Gly Leu Leu Val Tyr      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Th - #r Ala Thr Glu Pro        Pro                                                                                             405  - #               410  - #               415             - - Val Ile Thr Arg His Asn Tyr Asn Asn Ile Th - #r Leu Asn Thr Cys Val                  420      - #           425      - #           430                  - - Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gl - #y Phe Ile Thr Asn Val              435          - #       440          - #       445                      - - Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Al - #a Asp Ala Gly Leu Ala          450              - #   455              - #   460                          - - Ile Leu Asp Thr Ser Gly Ser Ile Asp Ile Ph - #e Val Val Gln Gly Glu      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cy - #s Glu Asp Val Asn        Gln                                                                                             485  - #               490  - #               495             - - Gln Phe Val Val Ser Gly Gly Lys Leu Val Gl - #y Ile Leu Thr Ser Arg                  500      - #           505      - #           510                  - - Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gl - #n Phe Tyr Ile Lys Ile              515          - #       520          - #       525                      - - Thr Asn Gly Thr Arg Arg Phe Arg Arg Ser Il - #e Thr Glu Asn Val Ala          530              - #   535              - #   540                          - - Asn Cys Pro Tyr Val Ser Tyr Gly Lys Phe Cy - #s Ile Lys Pro Asp Gly      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Ser Ile Ala Thr Ile Val Pro Lys Gln Leu Gl - #u Gln Phe Val Ala        Pro                                                                                              565 - #                570 - #                575            - - Leu Leu Asn Val Thr Glu Asn Val Leu Ile Pr - #o Asn Ser Phe Asn Leu                  580      - #           585      - #           590                  - - Thr Val Thr Asp Glu Tyr Ile Gln Thr Arg Me - #t Asp Lys Val Gln Ile              595          - #       600          - #       605                      - - Asn Cys Leu Gln Tyr Val Cys Gly Asn Ser Le - #u Asp Cys Arg Asp Leu          610              - #   615              - #   620                          - - Phe Gln Gln Tyr Gly Pro Val Cys Asp Asn Il - #e Leu Ser Val Val Asn      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Ser Ile Gly Gln Lys Glu Asp Met Glu Leu Le - #u Asn Phe Tyr Ser        Ser                                                                                             645  - #               650  - #               655             - - Thr Lys Pro Ala Gly Phe Asn Thr Pro Phe Le - #u Ser Asn Val Ser Thr                  660      - #           665      - #           670                  - - Gly Glu Phe Asn Ile Ser Leu Leu Leu Thr Th - #r Pro Ser Ser Pro Arg              675          - #       680          - #       685                      - - Arg Arg Ser Phe Ile Glu Asp Leu Leu Phe Th - #r Ser Val Glu Ser Val          690              - #   695              - #   700                          - - Gly Leu Pro Thr Asp Asp Ala Tyr Lys Asn Cy - #s Thr Ala Gly Pro Leu      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Gly Phe Leu Lys Asp Leu Ala Cys Ala Arg Gl - #u Tyr Asn Gly Leu        Leu                                                                                             725  - #               730  - #               735             - - Val Leu Pro Pro Ile Ile Thr Ala Glu Met Gl - #n Thr Leu Tyr Thr Ser                  740      - #           745      - #           750                  - - Ser Leu Val Ala Ser Met Ala Phe Gly Gly Il - #e Thr Ala Ala Gly Ala              755          - #       760          - #       765                      - - Ile Pro Phe Ala Thr Gln Leu Gln Ala Arg Il - #e Asn His Leu Gly Ile          770              - #   775              - #   780                          - - Thr Gln Ser Leu Leu Leu Lys Asn Gln Glu Ly - #s Ile Ala Ala Ser Phe      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Asn Lys Ala Ile Gly Arg Met Gln Glu Gly Ph - #e Arg Ser Thr Ser        Leu                                                                                             805  - #               810  - #               815             - - Ala Leu Gln Gln Ile Gln Asp Val Val Asn Ly - #s Gln Ser Ala Ile Leu                  820      - #           825      - #           830                  - - Thr Glu Thr Met Ala Ser Leu Asn Lys Asn Ph - #e Gly Ala Ile Ser Ser               835         - #        840         - #        845                     - - Val Ile Gln Glu Ile Tyr Gln Gln Leu Asp Al - #a Ile Gln Ala Asn Ala          850              - #   855              - #   860                          - - Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Se - #r Ser Leu Ser Val Leu      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Ala Ser Ala Lys Gln Ala Glu His Ile Arg Va - #l Ser Gln Gln Arg        Glu                                                                                             885  - #               890  - #               895             - - Leu Ala Thr Gln Lys Ile Asn Glu Cys Val Ly - #s Ser Gln Ser Ile Arg                  900      - #           905      - #           910                  - - Tyr Ser Phe Cys Gly Asn Gly Arg His Val Le - #u Thr Ile Pro Gln Asn              915          - #       920          - #       925                      - - Ala Pro Asn Gly Ile Val Phe Ile His Phe Se - #r Tyr Thr Pro Asp Ser          930              - #   935              - #   940                          - - Phe Val Asn Val Thr Ala Ile Val Gly Phe Cy - #s Val Lys Pro Ala Asn      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Ala Ser Gln Tyr Ala Ile Val Pro Ala Asn Gl - #y Arg Gly Ile Phe        Ile                                                                                             965  - #               970  - #               975             - - Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Ar - #g Asp Met Tyr Met Pro                  980      - #           985      - #           990                  - - Arg Ala Ile Thr Ala Gly Asp Ile Val Thr Le - #u Thr Ser Cys Gln Ala              995          - #       1000          - #      1005                     - - Asn Tyr Val Ser Val Asn Lys Thr Val Ile Th - #r Thr Phe Val Asp Asn          1010             - #   1015              - #  1020                         - - Asp Asp Phe Asp Phe Asn Asp Glu Leu Ser Ly - #s Trp Trp Asn Asp Thr      1025                1030 - #                1035 - #               1040        - - Lys His Glu Leu Pro Asp Phe Asp Lys Phe As - #n Tyr Thr Val Pro Ile                      1045 - #               1050  - #              1055             - - Leu Asp Ile Asp Ser Glu Ile Asp Arg Ile Gl - #n Gly Val Ile Gln Gly                  1060     - #           1065      - #          1070                 - - Leu Asn Asp Ser Leu Ile Asp Leu Glu Lys Le - #u Ser Ile Leu Lys Thr              1075         - #       1080          - #      1085                     - - Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Al - #a Ile Ala Phe Ala Thr          1090             - #   1095              - #  1100                         - - Ile Ile Phe Ile Leu Ile Leu Gly Trp Val Ph - #e Phe Met Thr Gly Cys      1105                1110 - #                1115 - #               1120        - - Cys Gly Cys Cys Cys Gly Cys Phe Gly Ile Me - #t Pro Leu Met Ser Lys                      1125 - #               1130  - #              1135             - - Cys Gly Lys Lys Ser Ser Tyr Tyr Thr Thr Ph - #e Asp Asn Asp Val Val                  1140     - #           1145      - #          1150                 - - Thr Glu Gln Asn Arg Pro Lys Lys Ser Val                                          1155         - #       1160                                            - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1846 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1846                                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:16:                       - - ATG TTG GTG AAG TCA CTG TTT CTA GTG ACC AT - #T TTG TTT GCA CTA TGT           48                                                                       Met Leu Val Lys Ser Leu Phe Leu Val Thr Il - #e Leu Phe Ala Leu Cys             1               5 - #                 10 - #                 15              - - AGT GCT AAT TTA TAT GAC AAC GAA TCT TTT GT - #G TAT TAC TAC CAG AGT           96                                                                       Ser Ala Asn Leu Tyr Asp Asn Glu Ser Phe Va - #l Tyr Tyr Tyr Gln Ser                        20     - #             25     - #             30                  - - GCT TTT AGG CCA GGA CAT GGT TGG CAT TTA CA - #T GGA GGT GCT TAT GCA          144                                                                       Ala Phe Arg Pro Gly His Gly Trp His Leu Hi - #s Gly Gly Ala Tyr Ala                    35         - #         40         - #         45                      - - GTA GTT AAT GTG TCT AGT GAA AAT AAT AAT GC - #A GGT ACT GCC CCA AGT          192                                                                       Val Val Asn Val Ser Ser Glu Asn Asn Asn Al - #a Gly Thr Ala Pro Ser                50             - #     55             - #     60                          - - TGC ACT GCT GGT GCT ATT GGC TAC AGT AAG AA - #T TTC AGT GCG GCC TCA          240                                                                       Cys Thr Ala Gly Ala Ile Gly Tyr Ser Lys As - #n Phe Ser Ala Ala Ser            65                 - # 70                 - # 75                 - # 80       - - GTA GCC ATG ACT GCA CCA CTA AGT GGT ATG TC - #A TGG TCT GCC TCA TCT          288                                                                       Val Ala Met Thr Ala Pro Leu Ser Gly Met Se - #r Trp Ser Ala Ser Ser                            85 - #                 90 - #                 95              - - TTT TGT ACA GCT CAC TGT AAT TTT ACT TCT TA - #T ATA GTG TTT GTT ACA          336                                                                       Phe Cys Thr Ala His Cys Asn Phe Thr Ser Ty - #r Ile Val Phe Val Thr                       100      - #           105      - #           110                  - - CAT TGT TTT AAG AGC GGA TCT AAT AGT TGT CC - #T TTG ACA GGT CTT ATT          384                                                                       His Cys Phe Lys Ser Gly Ser Asn Ser Cys Pr - #o Leu Thr Gly Leu Ile                   115          - #       120          - #       125                      - - CCA AGC GGT TAT ATT CGT ATT GCT GCT ATG AA - #A CAT GGA AGT CGT ACG          432                                                                       Pro Ser Gly Tyr Ile Arg Ile Ala Ala Met Ly - #s His Gly Ser Arg Thr               130              - #   135              - #   140                          - - CCT GGT CAC TTA TTT TAT AAC TTA ACA GTT TC - #T GTG ACT AAA TAT CCT          480                                                                       Pro Gly His Leu Phe Tyr Asn Leu Thr Val Se - #r Val Thr Lys Tyr Pro           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - AAG TTT AGA TCG CTA CAA TGT GTT AAT AAT CA - #T ACT TCT GTA TAT        TTA      528                                                                    Lys Phe Arg Ser Leu Gln Cys Val Asn Asn Hi - #s Thr Ser Val Tyr Leu                          165  - #               170  - #               175              - - AAT GGT GAC CTT GTT TTC ACA TCT AAC TAT AC - #T GAA GAT GTT GTA GCT          576                                                                       Asn Gly Asp Leu Val Phe Thr Ser Asn Tyr Th - #r Glu Asp Val Val Ala                       180      - #           185      - #           190                  - - GCA GGT GTC CAT TTT AAA AGT GGT GGA CCT AT - #A ACT TAT AAA GTT ATG          624                                                                       Ala Gly Val His Phe Lys Ser Gly Gly Pro Il - #e Thr Tyr Lys Val Met                   195          - #       200          - #       205                      - - AGA GAG GTT AAA GCC TTG GCT TAT TTT GTC AA - #T GGT ACT GCA CAT GAT          672                                                                       Arg Glu Val Lys Ala Leu Ala Tyr Phe Val As - #n Gly Thr Ala His Asp               210              - #   215              - #   220                          - - GTC ATT CTA TGT GAT GAC ACA CCT AGA GGT TT - #G TTA GCA TGC CAA TAT          720                                                                       Val Ile Leu Cys Asp Asp Thr Pro Arg Gly Le - #u Leu Ala Cys Gln Tyr           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - AAT ACT GGC AAT TTT TCA GAT GGC TTC TAT CC - #T TTT ACT AAT ACT        AGT      768                                                                    Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pr - #o Phe Thr Asn Thr Ser                          245  - #               250  - #               255              - - ATT GTT AAG GAT AAG TTT ATT GTT TAT CGT GA - #A AGT AGT GTC AAT ACT          816                                                                       Ile Val Lys Asp Lys Phe Ile Val Tyr Arg Gl - #u Ser Ser Val Asn Thr                       260      - #           265      - #           270                  - - ACT TTG ACA TTA ACT AAT TTC ACG TTT AGT AA - #T GAA AGT GGT GCC CCT          864                                                                       Thr Leu Thr Leu Thr Asn Phe Thr Phe Ser As - #n Glu Ser Gly Ala Pro                   275          - #       280          - #       285                      - - CCT AAT ACA GGT GGT GTT GAC AGT TTT ATT TT - #A TAC CAG ACA CAA ACA          912                                                                       Pro Asn Thr Gly Gly Val Asp Ser Phe Ile Le - #u Tyr Gln Thr Gln Thr               290              - #   295              - #   300                          - - GCT CAG AGT GGT TAT TAT AAT TTT AAT TTT TC - #A TTT CTG AGT AGT TTT          960                                                                       Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Se - #r Phe Leu Ser Ser Phe           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - GTT TAT AGG GAA AGT AAT TAT ATG TAT GGA TC - #T TAC CAT CCG GCT        TGT     1008                                                                    Val Tyr Arg Glu Ser Asn Tyr Met Tyr Gly Se - #r Tyr His Pro Ala Cys                          325  - #               330  - #               335              - - AGT TTT AGA CCT GAA ACC CTT AAT GGT TTG TG - #G TCT AAT TCC CTT TCT         1056                                                                       Ser Phe Arg Pro Glu Thr Leu Asn Gly Leu Tr - #p Ser Asn Ser Leu Ser                       340      - #           345      - #           350                  - - GTT TCA TTA ATA TAC GGT CCC ATT CAA GGT GG - #T TGT AAG CAA TCT GTA         1104                                                                       Val Ser Leu Ile Tyr Gly Pro Ile Gln Gly Gl - #y Cys Lys Gln Ser Val                   355          - #       360          - #       365                      - - TTT AAT GGT AAA GCA ACT TGT TGT TAT GCT TA - #T TCA TAC GGA GGA CCT         1152                                                                       Phe Asn Gly Lys Ala Thr Cys Cys Tyr Ala Ty - #r Ser Tyr Gly Gly Pro               370              - #   375              - #   380                          - - CGT GCT TGT AAA GGT GTC TAT AGA GGT GAG CT - #A ACA CAG CAT TTT GAA         1200                                                                       Arg Ala Cys Lys Gly Val Tyr Arg Gly Glu Le - #u Thr Gln His Phe Glu            385                 - #390                 - #395                 -         #400                                                                             - - TGT GGT TTG TTA GTT TAT GTT ACT AAG AGC GA - #T GGC TCC CGT ATA        CAA     1248                                                                    Cys Gly Leu Leu Val Tyr Val Thr Lys Ser As - #p Gly Ser Arg Ile Gln                          405  - #               410  - #               415              - - ACT GCA ACA CAA CCA CCT GTA TTA ACC CAA AA - #T TTT TAT AAT AAC ATC         1296                                                                       Thr Ala Thr Gln Pro Pro Val Leu Thr Gln As - #n Phe Tyr Asn Asn Ile                       420      - #           425      - #           430                  - - ACT TTA GGT AAG TGT GTT GAT TAT AAT GTT TA - #T GGT AGA ACT GGA CAA         1344                                                                       Thr Leu Gly Lys Cys Val Asp Tyr Asn Val Ty - #r Gly Arg Thr Gly Gln                   435          - #       440          - #       445                      - - GGT TTT ATT ACT AAT GTA ACT GAT TTA GCT AC - #T TCC CAT AAT TAC TTA         1392                                                                       Gly Phe Ile Thr Asn Val Thr Asp Leu Ala Th - #r Ser His Asn Tyr Leu               450              - #   455              - #   460                          - - GCG GAG GGA GGA TTA GCT ATT TTA GAT ACA TC - #T GGT GCC ATA GAC ATC         1440                                                                       Ala Glu Gly Gly Leu Ala Ile Leu Asp Thr Se - #r Gly Ala Ile Asp Ile           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - TTC GTT GTA CAA GGT GAA TAT GGC CCT AAC TA - #C TAT AAG GTT AAT        CTA     1488                                                                    Phe Val Val Gln Gly Glu Tyr Gly Pro Asn Ty - #r Tyr Lys Val Asn Leu                          485  - #               490  - #               495              - - TGT GAA GAT GTT AAC CAA CAG TTT GTA GTT TC - #T GGT GGT AAA TTA GTA         1536                                                                       Cys Glu Asp Val Asn Gln Gln Phe Val Val Se - #r Gly Gly Lys Leu Val                       500      - #           505      - #           510                  - - GGT ATT CTC ACT TCA CGT AAT GAA ACT GGT TC - #T CAG CCT CTT GAA AAC         1584                                                                       Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Se - #r Gln Pro Leu Glu Asn                   515          - #       520          - #       525                      - - CAG TTT TAC ATT AAG ATC ACT AAT GGA ACA CA - #T CGT TCT AGA CGT TCT         1632                                                                       Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr Hi - #s Arg Ser Arg Arg Ser               530              - #   535              - #   540                          - - GTT AAT GAA AAT GTT ACG AAT TGC CCT TAT GT - #T AGT TAT GGC AAG TTT         1680                                                                       Val Asn Glu Asn Val Thr Asn Cys Pro Tyr Va - #l Ser Tyr Gly Lys Phe           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - TGT ATA AAA CCT GAT GGT TCA GTT TCT CCT AT - #A GTA CCA AAA GAA        CTT     1728                                                                    Cys Ile Lys Pro Asp Gly Ser Val Ser Pro Il - #e Val Pro Lys Glu Leu                          565  - #               570  - #               575              - - GAA CAG TTT GTG GCA CCT TTA CTT AAT GTT AC - #T GAA AAT GTG CTC ATA         1776                                                                       Glu Gln Phe Val Ala Pro Leu Leu Asn Val Th - #r Glu Asn Val Leu Ile                       580      - #           585      - #           590                  - - CCT AAC AGT TTT AAC TTA ACT GTT ACA GAT GA - #G TAC ATA CAA ACG CGT         1824                                                                       Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Gl - #u Tyr Ile Gln Thr Arg                   595          - #       600          - #       605                      - - ATG GAT AAG GTC CAA ATT AGG A      - #                  - #                   1846                                                                     Met Asp Lys Val Gln Ile Arg                                                       610              - #   615                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 615 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:17:                    - - Met Leu Val Lys Ser Leu Phe Leu Val Thr Il - #e Leu Phe Ala Leu Cys        1               5 - #                 10 - #                 15              - - Ser Ala Asn Leu Tyr Asp Asn Glu Ser Phe Va - #l Tyr Tyr Tyr Gln Ser                   20     - #             25     - #             30                  - - Ala Phe Arg Pro Gly His Gly Trp His Leu Hi - #s Gly Gly Ala Tyr Ala               35         - #         40         - #         45                      - - Val Val Asn Val Ser Ser Glu Asn Asn Asn Al - #a Gly Thr Ala Pro Ser           50             - #     55             - #     60                          - - Cys Thr Ala Gly Ala Ile Gly Tyr Ser Lys As - #n Phe Ser Ala Ala Ser       65                 - # 70                 - # 75                 - # 80       - - Val Ala Met Thr Ala Pro Leu Ser Gly Met Se - #r Trp Ser Ala Ser Ser                       85 - #                 90 - #                 95              - - Phe Cys Thr Ala His Cys Asn Phe Thr Ser Ty - #r Ile Val Phe Val Thr                  100      - #           105      - #           110                  - - His Cys Phe Lys Ser Gly Ser Asn Ser Cys Pr - #o Leu Thr Gly Leu Ile              115          - #       120          - #       125                      - - Pro Ser Gly Tyr Ile Arg Ile Ala Ala Met Ly - #s His Gly Ser Arg Thr          130              - #   135              - #   140                          - - Pro Gly His Leu Phe Tyr Asn Leu Thr Val Se - #r Val Thr Lys Tyr Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Phe Arg Ser Leu Gln Cys Val Asn Asn Hi - #s Thr Ser Val Tyr        Leu                                                                                             165  - #               170  - #               175             - - Asn Gly Asp Leu Val Phe Thr Ser Asn Tyr Th - #r Glu Asp Val Val Ala                  180      - #           185      - #           190                  - - Ala Gly Val His Phe Lys Ser Gly Gly Pro Il - #e Thr Tyr Lys Val Met              195          - #       200          - #       205                      - - Arg Glu Val Lys Ala Leu Ala Tyr Phe Val As - #n Gly Thr Ala His Asp          210              - #   215              - #   220                          - - Val Ile Leu Cys Asp Asp Thr Pro Arg Gly Le - #u Leu Ala Cys Gln Tyr       225                 - #230                 - #235                 -         #240                                                                             - - Asn Thr Gly Asn Phe Ser Asp Gly Phe Tyr Pr - #o Phe Thr Asn Thr        Ser                                                                                             245  - #               250  - #               255             - - Ile Val Lys Asp Lys Phe Ile Val Tyr Arg Gl - #u Ser Ser Val Asn Thr                  260      - #           265      - #           270                  - - Thr Leu Thr Leu Thr Asn Phe Thr Phe Ser As - #n Glu Ser Gly Ala Pro              275          - #       280          - #       285                      - - Pro Asn Thr Gly Gly Val Asp Ser Phe Ile Le - #u Tyr Gln Thr Gln Thr          290              - #   295              - #   300                          - - Ala Gln Ser Gly Tyr Tyr Asn Phe Asn Phe Se - #r Phe Leu Ser Ser Phe      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Tyr Arg Glu Ser Asn Tyr Met Tyr Gly Se - #r Tyr His Pro Ala        Cys                                                                                             325  - #               330  - #               335             - - Ser Phe Arg Pro Glu Thr Leu Asn Gly Leu Tr - #p Ser Asn Ser Leu Ser                  340      - #           345      - #           350                  - - Val Ser Leu Ile Tyr Gly Pro Ile Gln Gly Gl - #y Cys Lys Gln Ser Val              355          - #       360          - #       365                      - - Phe Asn Gly Lys Ala Thr Cys Cys Tyr Ala Ty - #r Ser Tyr Gly Gly Pro          370              - #   375              - #   380                          - - Arg Ala Cys Lys Gly Val Tyr Arg Gly Glu Le - #u Thr Gln His Phe Glu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Cys Gly Leu Leu Val Tyr Val Thr Lys Ser As - #p Gly Ser Arg Ile        Gln                                                                                             405  - #               410  - #               415             - - Thr Ala Thr Gln Pro Pro Val Leu Thr Gln As - #n Phe Tyr Asn Asn Ile                  420      - #           425      - #           430                  - - Thr Leu Gly Lys Cys Val Asp Tyr Asn Val Ty - #r Gly Arg Thr Gly Gln              435          - #       440          - #       445                      - - Gly Phe Ile Thr Asn Val Thr Asp Leu Ala Th - #r Ser His Asn Tyr Leu          450              - #   455              - #   460                          - - Ala Glu Gly Gly Leu Ala Ile Leu Asp Thr Se - #r Gly Ala Ile Asp Ile      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Phe Val Val Gln Gly Glu Tyr Gly Pro Asn Ty - #r Tyr Lys Val Asn        Leu                                                                                             485  - #               490  - #               495             - - Cys Glu Asp Val Asn Gln Gln Phe Val Val Se - #r Gly Gly Lys Leu Val                   500     - #            505     - #            510                 - - Gly Ile Leu Thr Ser Arg Asn Glu Thr Gly Se - #r Gln Pro Leu Glu Asn              515          - #       520          - #       525                      - - Gln Phe Tyr Ile Lys Ile Thr Asn Gly Thr Hi - #s Arg Ser Arg Arg Ser          530              - #   535              - #   540                          - - Val Asn Glu Asn Val Thr Asn Cys Pro Tyr Va - #l Ser Tyr Gly Lys Phe      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Cys Ile Lys Pro Asp Gly Ser Val Ser Pro Il - #e Val Pro Lys Glu        Leu                                                                                             565  - #               570  - #               575             - - Glu Gln Phe Val Ala Pro Leu Leu Asn Val Th - #r Glu Asn Val Leu Ile                  580      - #           585      - #           590                  - - Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Gl - #u Tyr Ile Gln Thr Arg              595          - #       600          - #       605                      - - Met Asp Lys Val Gln Ile Arg                                                  610              - #   615                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2116 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:18:                       - - TATAATTATC TAGCAGACGC AGGTATGGCT ATTTTAGATA CATCTGGTTC CA -             #TAGACATC     60                                                                 - - TTTGTTGCAC AAGGTGAATA TGGCCTTACT TATTATAAGG CTAACCCTTG CG -            #AAGACGTC    120                                                                 - - AACCAGCAGT TTGTAGTTTC TGGTGGTAAA TTAGTAGGTA TTCTTACTTC AC -            #GTAATGAG    180                                                                 - - ACTGGTTCTC AGCTTCTTGA GAACCAGTTT TACATTAAAA TCACTAATGG AA -            #CACGTCGT    240                                                                 - - TCTAGACGTT CTATTACTGC AAATGTHACA AATYGCCCTT ATGTTAGCTA TG -            #GCAAGTTT    300                                                                 - - TGTCTAAAAC CTGATGGYTC AGYTTCTGYT ATAGCACCAC NNNNNNNNNN NN -            #NNNNNNNN    360                                                                 - - NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NN -            #NNNNNNNN    420                                                                 - - NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NN -            #NNNNNNNT    480                                                                 - - GTTTGTGGCA ATTCTCTGGA TTGTAGAAAG TTGYTTCAAC AATATGGGCC TG -            #TTTGBGAC    540                                                                 - - AACATATTGT CTGTGGTAAA TAGTGTTGGT CAAAAAGAAG ATATGGAACT TC -            #UAAATCTC    600                                                                 - - TATTCTTCTA CTAAACCATC TGGCTTTAAT ACACCAGTTT TTAGTAATCT YA -            #GCACTGGC    660                                                                 - - GATTTYAATA TTTCTCTTYT GGTTGACACC TCCAGTAGTA CTACTGGGCG CT -            #CTTTTATT    720                                                                 - - GAAGATCTTT TATTTACAAG TGTTGAATCT GTTGGATTAC CAACAGATGA AG -            #CTTATAAA    780                                                                 - - AAGTGCACTG CAGGACCTTT AGGCTTCCTT AAGGACCTBG CGTGTGCTCG TG -            #AATATAAT    840                                                                 - - GGCTTGCTTG YNNNNNNCCC TATTATAACA GCAGAAATGC AAACCTTGTA TA -            #CTAGTTCT    900                                                                 - - TTAGTAGCTT CTATGGCTTT TGGTGGGATT ACTGCAGCTG GTGCTATACC TT -            #TTGCCACA    960                                                                 - - CAACTGCAGG CTAGAATTAA TCACTTGGGT ATTACCCAGT CACTTTTGCA GA -            #AAAATCAA   1020                                                                 - - GAAAAAATTG CTGCCTCCTT TAATAAGGCC ATTGGCCATA TGCAGGAAGG TT -            #TTAGAAGT   1080                                                                 - - ACATCTCTAG CATTACAACA AGTYCAMGAT GTTGTTAATA AGCAGAGTGC TA -            #TTCTTACT   1140                                                                 - - GAGACTATGG CATCACTTAA TAAAAATTTK GGTGCTATTT CTTCTGTGAT TC -            #AAGATATC   1200                                                                 - - TACCAGCAAC TTGACGCCAT ACAAGCAAAT GCTCAAGTGG ATCGTCTTAT AA -            #CTGGTAGA   1260                                                                 - - TTGTCATCAC TTTCTGTTTT AGCATCTGCT AAGCAGGCGG AGTATATTAG AG -            #TGTCACAA   1320                                                                 - - CAGCGTGAGT TAGCTACTCA GAAAATTAAT GAGTGTGTTA AATCACAGTC TA -            #TTAGGTAC   1380                                                                 - - TCCTTTTGTG GTAATGGACG ACACGTTCTA ACTATACCGC AAAATGCACC TA -            #ATGGTATA   1440                                                                 - - GTGTTTATAC ACTTTACTTA TACTCCAGAG AGTTTTGKTA ATGTTACTGC AA -            #TAGTGGGT   1500                                                                 - - TTTTGTAARG CCGCTAATGC TAGTCAGTAT GCAATAGTGC CTGCTAATGG CA -            #GAGGTATT   1560                                                                 - - TCTATACAAG TTAATGGTAG TCACTACATC ACTGCACGAG ATATGTATAT GC -            #CAAGAGAT   1620                                                                 - - ATTACTGCAG GAGATATAGT TACGCTTACT TCTTGTCAAG CAAATTATGT AA -            #GTGTAMMT   1680                                                                 - - AAGACCGTCA TTACYACATT HGTAGACAAT GATGATTTTG ATTTTGATGA CG -            #AATTGTCA   1740                                                                 - - AAATGGTGGA ATGATACTAA GCATGAGCTA CCAGACTTTG ACGAATTCAA TT -            #ACACAGTA   1800                                                                 - - CCTATACTTG ACATTGGTAG TGAAATTGAT CGTATTCAAG GCGTTATACA GG -            #GCCTTAAT   1860                                                                 - - GACTCTCTAA TAGACCTTGA AACACTATCA ATACTCAAAA CTTATATTAA GT -            #GGCCTTGG   1920                                                                 - - TATGTGTGGT TAGCCATAGC TTTTGSCACT ATTATCTTCA TCCTAATATT AG -            #GGTGGGTG   1980                                                                 - - TTTTTCATGA CTGGTTGTTG TGGTTGTTGT TGTGGATGCT TTGGCATTAT TC -            #CTCTAATG   2040                                                                 - - AGCAAGTGTG GTAAGAAATC TTCTTATTAC ACGACTTTGG ATAATGATGT GG -            #TAACTGAA   2100                                                                 - - CAAWACAGAC CYAAAA             - #                  - #                      - #  2116                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 705 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:19:                       - - Tyr Asn Tyr Leu Ala Asp Ala Gly Met Ala Il - #e Leu Asp Thr Ser Gly       1               5  - #                 10 - #                 15              - - Ser Ile Asp Ile Phe Val Ala Gln Gly Glu Ty - #r Gly Leu Thr Tyr Tyr                  20      - #            25      - #            30                   - - Lys Ala Asn Pro Cys Glu Asp Val Asn Gln Gl - #n Phe Val Val Ser Gly              35          - #        40          - #        45                       - - Gly Lys Leu Val Gly Ile Leu Thr Ser Arg As - #n Glu Thr Gly Ser Gln          50              - #    55              - #    60                           - - Leu Leu Glu Asn Gln Phe Tyr Ile Lys Ile Th - #r Asn Gly Thr Arg Arg      65                  - #70                  - #75                  - #80        - - Ser Arg Arg Ser Ile Thr Ala Asn Val Thr As - #n Xaa Pro Tyr Val Ser                      85  - #                90  - #                95               - - Tyr Gly Lys Phe Cys Leu Lys Pro Asp Gly Se - #r Xaa Ser Xaa Ile Ala                  100      - #           105      - #           110                  - - Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa              115          - #       120          - #       125                      - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa          130              - #   135              - #   140                          - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Cys Gly Asn Ser Leu Asp Cys Arg Lys Le - #u Xaa Gln Gln Tyr        Gly                                                                                             165  - #               170  - #               175             - - Pro Val Xaa Asp Asn Ile Leu Ser Val Val As - #n Ser Val Gly Gln Lys                  180      - #           185      - #           190                  - - Glu Asp Met Glu Leu Leu Asn Leu Tyr Ser Se - #r Thr Lys Pro Ser Gly              195          - #       200          - #       205                      - - Phe Asn Thr Pro Val Phe Ser Asn Leu Ser Th - #r Gly Asp Phe Asn Ile          210              - #   215              - #   220                          - - Ser Leu Leu Val Asp Thr Ser Ser Ser Thr Th - #r Gly Arg Ser Phe Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Va - #l Gly Leu Pro Thr        Asp                                                                                             245  - #               250  - #               255             - - Glu Ala Tyr Lys Lys Cys Thr Ala Gly Pro Le - #u Gly Phe Leu Lys Asp                  260      - #           265      - #           270                  - - Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Le - #u Xaa Xaa Xaa Pro Ile              275          - #       280          - #       285                      - - Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Se - #r Ser Leu Val Ala         Ser                                                                                 290              - #   295              - #   300                          - - Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Al - #a Ile Pro Phe Ala        Thr                                                                             305                 3 - #10                 3 - #15                 3 -     #20                                                                             - - Gln Leu Gln Ala Arg Ile Asn His Leu Gly Il - #e Thr Gln Ser Leu Leu                        - #   325              - #   330              - #          335                                                                              - - Gln Lys Asn Gln Glu Lys Ile Ala Ala Ser Ph - #e Asn Lys Ala Ile        Gly                                                                                         340      - #           345      - #           350                  - - His Met Gln Glu Gly Phe Arg Ser Thr Ser Le - #u Ala Leu Gln Gln        Val                                                                                     355          - #       360          - #       365                      - - Xaa Asp Val Val Asn Lys Gln Ser Ala Ile Le - #u Thr Glu Thr Met        Ala                                                                                 370              - #   375              - #   380                          - - Ser Leu Asn Lys Asn Xaa Gly Ala Ile Ser Se - #r Val Ile Gln Asp        Ile                                                                             385                 3 - #90                 3 - #95                 4 -     #00                                                                             - - Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Al - #a Gln Val Asp Arg Leu                      405  - #               410  - #               415              - - Ile Thr Gly Arg Leu Ser Ser Leu Ser Val Le - #u Ala Ser Ala Lys Gln                  420      - #           425      - #           430                  - - Ala Glu Tyr Ile Arg Val Ser Gln Gln Arg Gl - #u Leu Ala Thr Gln Lys              435          - #       440          - #       445                      - - Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Ar - #g Tyr Ser Phe Cys Gly          450              - #   455              - #   460                          - - Asn Gly Arg His Val Leu Thr Ile Pro Gln As - #n Ala Pro Asn Gly Ile      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Val Phe Ile His Phe Thr Tyr Thr Pro Glu Se - #r Phe Xaa Asn Val        Thr                                                                                             485  - #               490  - #               495             - - Ala Ile Val Gly Phe Cys Lys Ala Ala Asn Al - #a Ser Gln Tyr Ala Ile                  500      - #           505      - #           510                  - - Val Pro Ala Asn Gly Arg Gly Ile Ser Ile Gl - #n Val Asn Gly Ser His              515          - #       520          - #       525                      - - Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Ar - #g Asp Ile Thr Ala Gly          530              - #   535              - #   540                          - - Asp Ile Val Thr Leu Thr Ser Cys Gln Ala As - #n Tyr Val Ser Val Xaa      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Lys Thr Val Ile Thr Thr Xaa Val Asp Asn As - #p Asp Phe Asp Phe        Asp                                                                                             565  - #               570  - #               575             - - Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Ly - #s His Glu Leu Pro Asp                  580      - #           585      - #           590                  - - Phe Asp Glu Phe Asn Tyr Thr Val Pro Ile Le - #u Asp Ile Gly Ser Glu              595          - #       600          - #       605                      - - Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Le - #u Asn Asp Ser Leu Ile          610              - #   615              - #   620                          - - Asp Leu Glu Thr Leu Ser Ile Leu Lys Thr Ty - #r Ile Lys Trp Pro Trp      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Tyr Val Trp Leu Ala Ile Ala Phe Xaa Thr Il - #e Ile Phe Ile Leu        Ile                                                                                             645  - #               650  - #               655             - - Leu Gly Trp Val Phe Phe Met Thr Gly Cys Cy - #s Gly Cys Cys Cys Gly                  660      - #           665      - #           670                  - - Cys Phe Gly Ile Ile Pro Leu Met Ser Lys Cy - #s Gly Lys Lys Ser Ser              675          - #       680          - #       685                      - - Tyr Tyr Thr Thr Leu Asp Asn Asp Val Val Th - #r Glu Gln Xaa Arg Pro          690              - #   695              - #   700                          - - Lys                                                                       - - 705                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:20:                       - - GAATTCGAGC TCGCCCGGGG ATCCTCTAGA GTCGAC      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 13..57                                                 - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:21:                       - - CACAGCTCAA CA ATG AAG TGG GCA ACG TGG ATC GAT - # CCC GTC GTT TTA             48                                                                                     Met Lys Tr - #p Ala Thr Trp Ile Asp Pro Val Val Leu                             1  - #             5     - #             10                    - - CAA CGT CGT              - #                  - #                       - #         57                                                                  Gln Arg Arg                                                                            15                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:22:                    - - Met Lys Trp Ala Thr Trp Ile Asp Pro Val Va - #l Leu Gln Arg Arg           1               5 - #                 10 - #                 15              - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:23:                       - - ACTCGGGCAG CGTTGGGTCC TGGGACTCTA GAGGATCGAT CCCCTATGGC GA - #TCATC            57                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:24:                       - - GCGCCCACGT GGCCTGGTAC AATTCGAGCT CGCCCGGGGA TCCTCTAGAG TC -             #GACTCTAG     60                                                                 - - AGGATCGATC CTCTAGAGTC GGCGGGACGA GCCCGCGAT      - #                      - #    99                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:25:                       - - TCCACAGGAC CTGCAGCGAC CCGCTTAACA GCGTCAACAG CGTGCCGCAG AT - #CGGGG            57                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:26:                       - - GTTGATCCCG GGAGATGGGG GAGGCTAACT GAAAC       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:27:                       - - GCTCATGGTG GCCCCCGGGC GGTTCAACGA GGGCCAGTAC CGGCGCCTGG TG -             #TCCGTCGA     60                                                                 - - CCTGCAGGTC GACTCTAGAG GATCCCCGGG CGAGCTCGAA TTC    - #                      - #103                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:28:                       - - GAATTCGAGC TCGCCCGGGG ATCCTCTAGA GTCGACGTCT GGGGCGCGGG GG -             #TGGTGCTC     60                                                                 - - TTCGAG                 - #                  - #                  -     #           66                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 16..66                                                 - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:29:                       - - CTCCACAGCT CAACA ATG AAG TGG GCA ACG TGG ATC - #GAT CCC GTC GTT TTA          51                                                                                          - #Met Lys Trp Ala Thr Trp Ile Asp Pro Val V - #al Leu                        - # 1               5  - #                10                 - - CAA CGT CGT GAC TGG           - #                  - #                      - #    66                                                                  Gln Arg Arg Asp Trp                                                                    15                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:30:                    - - Met Lys Trp Ala Thr Trp Ile Asp Pro Val Va - #l Leu Gln Arg Arg Asp        1               5 - #                 10 - #                 15              - - Trp                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 132 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..93                                                  - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:31:                       - - GAC GAC TCC TGG AGC CCG TCA GTA TCG GCG GA - #A ATC CAG CTG AGC GCC           48                                                                       Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Gl - #u Ile Gln Leu Ser Ala             1               5 - #                 10 - #                 15              - - GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT CA - #A AAA GAT CTA GAA               - #93                                                                    Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gl - #n Lys Asp Leu Glu                            20     - #             25     - #             30                  - - TAAGCTAGAG GATCGATCCC CTATGGCGAT CATCAGGGC      - #                      - #   132                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:32:                    - - Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Gl - #u Ile Gln Leu Ser Ala        1               5 - #                 10 - #                 15              - - Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gl - #n Lys Asp Leu Glu                       20     - #             25     - #             30                  - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:33:                       - - AACGAGGGCC AGTACCGGCG CCTGGTGTCC GTCGACTCTA GAGGATCCCC GG -             #GCGAGCTC     60                                                                 - - GAATTC                 - #                  - #                  -     #           66                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:34:                       - - CAGGTCGAAG CTTGGGCGCT GCCTATGTAG TGAAATCTAT ACTGGGATTT AT -             #CATAACTA     60                                                                 - - GTTTA                 - #                  - #                  -      #            65                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:35:                       - - AATAATCTAT CACTTTGTCA TGGAGATGCC CAAGCTTCGA CGACTCCCTT GG -            #CCATGATG     60                                                                 - - AATGG                 - #                  - #                  -      #            65                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:36:                       - - TATACCAGCT ACGGCGCTAG CATTCATGGT ATCCCGTGAT TGCTCGATGC TT -            #TCCTTCTG     60                                                                 - - AATTC                 - #                  - #                  -      #            65                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:37:                       - - AAGCTTGGCC TCGTCGTTAA TTAACCCAAT TCGAGCTCGC CCAGCTTGGG CT -            #GCAGGTCG     60                                                                 - - GGAAC                 - #                  - #                  -      #            65                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:38:                       - - TGTTTCAGTT AGCCTCCCCC ATCTCCCGAC TCTAGAGGAT CTCGACATAG CG -            #AATACATT     60                                                                 - - TATGG                 - #                  - #                  -      #            65                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 130 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:39:                       - - AACGTATATA TTTTTCACGA CGTAGACCAC TATTGCCATG GACTCTAGAG GA -            #TCGGGTAC     60                                                                 - - CGAGCTCGAA TTGGGAAGCT TGTCGACTTA ATTAAGCGGC CGCGTTTAAA CG -            #GCCCTCGA    120                                                                 - - GGCCAAGCTT                - #                  - #                      - #       130                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:40:                       - - GTCGACGTCT GGGGCGCGGG GGTGGTGCTC TTCGAGACGC TGCCTACCCC AA -             #GACGATCG     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:41:                       - - AGCTCAACAA TGAAGTGGGC AACGTGGATC GATCCCGTCG TTTTACAACG TC -            #GTGACTGG     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:42:                       - - GAGCCCGTCA GTATCGGCGG AAATCCAGCT GAGCGCCGGT CGCTACCATT AC -            #CAGTTGGT     60                                                                 - - GTTGGTCTGG TGTCAAAAAG ATCCGGACCG CGCCGTTAGC CAAGTTGCGT TA -            #GAGAATGA    120                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:43:                       - - ACACAGTCAC ACTCATGGGG GCCGAAGGCA GAATTCGTAA TCATGGTCAT AG -            #CTGTTTCC     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:44:                       - - AAACCTGTCG TGCCAGCGAG CTCGGGATCC TCTAGAGGAT CCCCGGGCCC CG -            #CCCCCTGC     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:45:                       - - TCGTCCACAC GGAGCGCGGC TGCCGACACG GATCCCGGTT GGCGCCCTCC AG -            #GTGCAGGA     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:46:                       - - AACCCCCCCC CCCCCCCCCC CCCCCCCCTG CAGGCATCGT GGTGTCACGC TC -            #GTCGTTTG     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:47:                       - - TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TCGGATCCTC TA -            #GAGTCGAC     60                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2681 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 146..481                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: complement - #(602..1402)                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1599..2135                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: complement - #(2308..2634)                             - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:48:                       - - TTTATCGGAC CTTGGGTATT CAGGGGAACC CATCTGGTTG AAATGCATCC GA -            #CCCTGCAC     60                                                                 - - TTGATCCTGG TTACCCCGAC CCAANTTTTA AGCCGGCTGG CGCGGTCCCT AG -            #ATAACCCC    120                                                                 - - CCGCTTAAAA CTAGCCCCAA TATTGATGTG CAGATATAAC ACAGNNANCC GA -            #TCAATGGA    180                                                                 - - AGACATGCTA CGGCGGTCAT CTCCCGAAGA CATCACCGAT TCCCTAACAA TG -            #TGCCTGAT    240                                                                 - - TATGTTATCG CGCATTCGTC GTACCATGCG CACCGCAGGA AATAAATATA GC -            #TATATGAT    300                                                                 - - AGATCCAATG AATCGTATGT CTAATTACAC TCCAGGCGAA TGTATGACAG GT -            #ATATTGCG    360                                                                 - - ATATATTGAC GAACATGCTA GAAGGTGTCC TGATCACATA TGTAATTTGT AT -            #ATCACATG    420                                                                 - - TACACTTATG CCGATGTATG TGCACGGGCG ATATTTCTAT TGTAATTCAT TT -            #TTTTGKTA    480                                                                 - - GTAAACTACC ACAGGCTGTC CGGAAATCTA AGTTAATGAA TAAAGTAGAT GG -            #TTAATACT    540                                                                 - - CATTGCTTAG AATTGGACTA CTTTTAATYC TCTTTAATGT TCGTATTAAA TA -            #AAAACATC    600                                                                 - - TTTAATAAAC TTCAGCCTCT TCGCTTATTG TAGAAATTGA GTATTCAMAA TC -            #ATGTTCAA    660                                                                 - - AGCCGTCTTC GGAGAGTGTA CTCGCCACGG TGGTTGGAAC ATCACTATGT CT -            #ACACGTCA    720                                                                 - - AATTTAAGCA CGTCAGGTCT GTCGAGGACA AGAAATGGTT AACTAGTGTT TC -            #AATTATTC    780                                                                 - - TTATAAACGT TAAGCATTGT AAGCCCCCCG GCCGTCCGCA GCAACAATTT AC -            #TAGTATGC    840                                                                 - - CGTGGGCTCC GGGACTATCA CGGATGTCCA ATTCGCACAT GCATATAATT TT -            #TCTAGGGT    900                                                                 - - CTCTCATTTC GAGAAATCTT CGGGGATCCA TCAGCAATGC GGGCTGTAGT CC -            #CGATTCCC    960                                                                 - - GTTTCAAATG AAGGTGCTCC AACACGGTCT TCAAAGCAAC CGGCATACCA GC -            #AAACACAG   1020                                                                 - - ACTGCAACTC CCCGCTGCAA TGATTGGTTA TAAACAGTAA TCTGTCTTCT GG -            #AAGTATAT   1080                                                                 - - TTCGCCCGAC AATCCACGGC GCCCCCAAAG TTAAAAACCA TCCATGTGTA TT -            #TGCGTCTT   1140                                                                 - - CTCTGTTAAA AGAATATTGA CTGGCATTTT CCCGTTGACC GCCAGATATC CA -            #AAGTACAG   1200                                                                 - - CACGATGTTG CACGGACGAC TTTGCAGTCA CCAGCCTTCC TTTCCACCCC CC -            #CACCAACA   1260                                                                 - - AAATGTTTAT CGTAGGACCC ATATCCGTAA TAAGGATGGG TCTGGCAGCA AC -            #CCCATAGG   1320                                                                 - - CGCCTCGGCG TGGTAGTTCT CGAGGATACA TCCAAAGAGG TTGAGTATTC TC -            #TCTACACT   1380                                                                 - - TCTTGTTAAA TGGAAAGTGC ATTTGCTTGT TCTTACAATC GGCCCGAGTC TC -            #GTTCACAG   1440                                                                 - - CGCCTCGTTC ACACTTAAAC CACAAATAGT CTACAGGCTA TATGGGAGCC AG -            #ACTGAAAC   1500                                                                 - - TCACATATGA CTAATATTCG GGGGTGTTAG TCACGTGTAG CCCATTGTGT GC -            #ATATAACG   1560                                                                 - - ATGTTGGACG CGTCCTTATT CGCGGTGTAC TTGATACTAT GGCAGCGAGC AT -            #GGGATATT   1620                                                                 - - CATCCTCGTC ATCGTTAACA TCTCTACGGG TTCAGAATGT TTGGCATGTC GT -            #CGATCCTT   1680                                                                 - - TGCCCATCGT TGCAAATTAC AAGTCCGATC GCCATGACCG CGATAAGCCT GT -            #ACCATGTG   1740                                                                 - - GCATTAGGGT GACATCTCGA TCATACATTA TAAGACCAAC GTGCGAGTCT TC -            #CAAAGACC   1800                                                                 - - TGCACGCCTT CTTCTTCGGA TTGTCAACGG GTTCTTCAGA ATCTATGCCC AT -            #ATCTGGCG   1860                                                                 - - TTGAGACCAT TGTGCGTTTA ATGAACAATA AAGCGGCATG CCATGGAAAG GA -            #GGGCTGCA   1920                                                                 - - GATCTCCATT TTCTCACGCC ACTATCCTGG ACGCTGTAGA CGATAATTAT AC -            #CATGAATA   1980                                                                 - - TAGAGGGGGT ATGTTTCCAC TGCCACTGTG ATGATAAGTT TTCTCCAGAT TG -            #TTGGATAT   2040                                                                 - - CTGCATTTTC TGCTGCCGAA CAAACTTCAT CGCTATGCAA AGAGATGCGT GT -            #GTACACGC   2100                                                                 - - GCCGGTGGAG TATACGGGAA ACTAAATGTT CATAGAGGTC TTTGGGCTAT AT -            #GTTATTAA   2160                                                                 - - ATAAAATAAT TGACCAGTGA ACAATTTGTT TAATGTTAGT TTATTCAATG CA -            #TTGGTTGC   2220                                                                 - - AAATATTCAT TACTTCTCCA ATCCCAGGTC ATTCTTTAGC GAGATGATGT TA -            #TGACATTG   2280                                                                 - - CTGTGAAAAT TACTACAGGA TATATTTTTA AGATGCAGGA GTAACAATGT GC -            #ATAGTAGG   2340                                                                 - - CGTAGTTATC GCAGACGTGC AACGCTTCGC ATTTGAGTTA CCGAAGTGCC CA -            #ACAGTGCT   2400                                                                 - - GCGGTTATGG TTTATGCGCA CAGAATCCAT GCATGTCCTA ATTGAACCAT CC -            #GATTTTTC   2460                                                                 - - TTTTAATCGC GATCGATGTT TGGGCAACTG CGTTATTTCA GATCTAAAAA AT -            #TTACCCTY   2520                                                                 - - TATGACCATC ACATCTCTCT GGYTCATACC CCGCTTGGGN TAAGATATCA TG -            #TAGATTCC   2580                                                                 - - GCCCCTAAGA AATTGCAAAC TAACATNATT GNCGGGTTCC ATATACAATC CC -            #ATCTTGTC   2640                                                                 - - CNCTCGAAAT TACAAACTCG CGCAATAGAC CCCCGTACAT T    - #                      - # 2681                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 111 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:49:                       - - Met Cys Arg Tyr Asn Thr Xaa Xaa Arg Ser Me - #t Glu Asp Met Leu Arg      1               5   - #                10  - #                15               - - Arg Ser Ser Pro Glu Asp Ile Thr Asp Ser Le - #u Thr Met Cys Leu Ile                  20      - #            25      - #            30                   - - Met Leu Ser Arg Ile Arg Arg Thr Met Arg Th - #r Ala Gly Asn Lys Tyr              35          - #        40          - #        45                       - - Ser Tyr Met Ile Asp Pro Met Asn Arg Met Se - #r Asn Tyr Thr Pro Gly          50              - #    55              - #    60                           - - Glu Cys Met Thr Gly Ile Leu Arg Tyr Ile As - #p Glu His Ala Arg Arg      65                  - #70                  - #75                  - #80        - - Cys Pro Asp His Ile Cys Asn Leu Tyr Ile Th - #r Cys Thr Leu Met Pro                       85 - #                 90 - #                 95              - - Met Tyr Val His Gly Arg Tyr Phe Tyr Cys As - #n Ser Phe Phe Xaa                      100      - #           105      - #           110                  - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 266 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:50:                       - - Met His Phe Pro Phe Asn Lys Lys Cys Arg Gl - #u Asn Thr Gln Pro Leu      1               5   - #                10  - #                15               - - Trp Met Tyr Pro Arg Glu Leu Pro Arg Arg Gl - #y Ala Tyr Gly Val Ala                  20      - #            25      - #            30                   - - Ala Arg Pro Ile Leu Ile Thr Asp Met Gly Pr - #o Thr Ile Asn Ile Leu              35          - #        40          - #        45                       - - Leu Val Gly Gly Trp Lys Gly Arg Leu Val Th - #r Ala Lys Ser Ser Val          50              - #    55              - #    60                           - - Gln His Arg Ala Val Leu Trp Ile Ser Gly Gl - #y Gln Arg Glu Asn Ala      65                  - #70                  - #75                  - #80        - - Ser Gln Tyr Ser Phe Asn Arg Glu Asp Ala As - #n Thr His Gly Trp Phe                      85  - #                90  - #                95               - - Leu Thr Leu Gly Ala Pro Trp Ile Val Gly Ar - #g Asn Ile Leu Pro Glu                  100      - #           105      - #           110                  - - Asp Arg Leu Leu Phe Ile Thr Asn His Cys Se - #r Gly Glu Leu Gln Ser              115          - #       120          - #       125                      - - Val Phe Ala Gly Met Pro Val Ala Leu Lys Th - #r Val Leu Glu His Leu          130              - #   135              - #   140                          - - His Leu Lys Arg Glu Ser Gly Leu Gln Pro Al - #a Leu Leu Met Asp Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Arg Phe Leu Glu Met Arg Asp Pro Arg Ly - #s Ile Ile Cys Met        Cys                                                                                             165  - #               170  - #               175             - - Glu Leu Asp Ile Arg Asp Ser Pro Gly Ala Hi - #s Gly Ile Leu Val Asn                  180      - #           185      - #           190                  - - Cys Cys Cys Gly Arg Pro Gly Gly Leu Gln Cy - #s Leu Thr Phe Ile Arg              195          - #       200          - #       205                      - - Ile Ile Glu Thr Leu Val Asn His Phe Leu Se - #r Ser Thr Asp Leu Thr          210              - #   215              - #   220                          - - Cys Leu Asn Leu Thr Cys Arg His Ser Asp Va - #l Pro Thr Thr Val Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Thr Leu Ser Glu Asp Gly Phe Glu His As - #p Xaa Glu Tyr Ser        Ile                                                                                              245 - #                250 - #                255            - - Ser Thr Ile Ser Glu Glu Ala Glu Val Tyr                                              260      - #           265                                         - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 178 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:51:                       - - Met Ala Ala Ser Met Gly Tyr Ser Ser Ser Se - #r Ser Leu Thr Ser Leu      1               5   - #                10  - #                15               - - Arg Val Gln Asn Val Trp His Val Val Asp Pr - #o Leu Pro Ile Val Ala                  20      - #            25      - #            30                   - - Asn Tyr Lys Ser Asp Arg His Asp Arg Asp Ly - #s Pro Val Pro Cys Gly              35          - #        40          - #        45                       - - Ile Arg Val Thr Ser Arg Ser Tyr Ile Ile Ar - #g Pro Thr Cys Glu Ser          50              - #    55              - #    60                           - - Ser Lys Asp Leu His Ala Phe Phe Phe Gly Le - #u Ser Thr Gly Ser Ser      65                  - #70                  - #75                  - #80        - - Glu Ser Met Pro Ile Ser Gly Val Glu Thr Il - #e Val Arg Leu Met         Asn                                                                                             85  - #                90  - #                95              - - Asn Lys Ala Ala Cys His Gly Lys Glu Gly Cy - #s Arg Ser Pro Phe Ser                  100      - #           105      - #           110                  - - His Ala Thr Ile Leu Asp Ala Val Asp Asp As - #n Tyr Thr Met Asn Ile              115          - #       120          - #       125                      - - Glu Gly Val Cys Phe His Cys His Cys Asp As - #p Lys Phe Ser Pro Asp          130              - #   135              - #   140                          - - Cys Trp Ile Ser Ala Phe Ser Ala Ala Glu Gl - #n Thr Ser Ser Leu Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Glu Met Arg Val Tyr Thr Arg Arg Trp Se - #r Ile Arg Glu Thr        Lys                                                                                             165  - #               170  - #               175             - - Cys Ser                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:52:                       - - Met Gly Leu Tyr Met Glu Pro Xaa Asn Xaa Va - #l Ser Leu Gln Phe Leu      1               5   - #                10  - #                15               - - Arg Gly Gly Ile Tyr Met Ile Ser Xaa Pro Ly - #s Arg Gly Met Xaa Gln                  20      - #            25      - #            30                   - - Arg Asp Val Met Val Ile Xaa Gly Lys Phe Ph - #e Arg Ser Glu Ile Thr              35          - #        40          - #        45                       - - Gln Leu Pro Lys His Arg Ser Arg Leu Lys Gl - #u Lys Ser Asp Gly Ser          50              - #    55              - #    60                           - - Ile Arg Thr Cys Met Asp Ser Val Arg Ile As - #n His Asn Arg Ser Thr      65                  - #70                  - #75                  - #80        - - Val Gly His Phe Gly Asn Ser Asn Ala Lys Ar - #g Cys Thr Ser Ala Ile                      85  - #                90  - #                95               - - Thr Thr Pro Thr Met His Ile Val Thr Pro Al - #a Ser                                  100      - #           105                                         - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA Oligonucleotide P - #rimer                    - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:53:                       - - CTCGCTCGCC CATGATCATT AAGCAAGAAT TCCGTCG      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA Oligonucleotide P - #rimer                    - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:54:                       - - CTGGTTCGGC CCATGATCAG ATGACAAACC TGCAAGATC      - #                      - #    39                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:55:                       - - CTCGGCGTGG TAGTTCTCGA GGCCTTAATT AAGGCCCTCG AGGATACATC CA - #AAGAG            57                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:56:                       - - CGGCGTGGTA GTTCTCGAGG CCTTAAGCGG CCGCTTAAGG CCCTCGAGGA TA -             #CATCCAAA     60                                                                 - - GAG                  - #                  - #                  - #                 63                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:57:                       - - CGCAGGATCC GGGGCGTCAG AGGCGGGCGA GGTG       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:58:                       - - GAGCGGATCC TGCAGGAGGA GACACAGAGC TG       - #                  - #              32                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:59:                       - - TGTAGAGATC TGGCTAAGTG CGCGTGTTGC CTG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:60:                       - - TGTACAGATC TCACCATGGC TGTGCCTGCA AGC       - #                  - #             33                                                                    __________________________________________________________________________

What is claimed is:
 1. A recombinant chimeric virus comprising a uniquelong viral genomic region which naturally occurs in a herpesvirus ofturkeys and a unique short viral genomic region which naturally occursin a Marek's disease virus.
 2. The recombinant chimeric virus of claim1, wherein a foreign DNA is inserted within a non essential region ofthe chimeric virus such that the foreign DNA expressed in a host cell.3. The recombinant chimeric virus of claim 2, wherein the non-essentialregion is an EcoR1 #9 fragment of the unique long viral genomic regionwhich naturally occurs in a herpesvirus of turkeys.
 4. The recombinantchimeric virus of claim 2, wherein the foreign DNA encodes apolypeptide.
 5. The recombinant chimeric virus of claim 2, wherein theforeign DNA encodes a cytokine.
 6. The recombinant chimeric virus ofclaim 5, wherein the cytokine is a chicken mylemonocytic growth factor(cMGF) or a chicken interferon (cIFN).
 7. The recombinant chimeric virusof claim 2, wherein the foreign DNA encodes E. coli beta-galactosidase.8. The recombinant chimeric virus of claim 4, wherein the polypeptide isobtained from Marek's Disease Virus, Newcastle Disease Virus, InfectiousLaryngotracheitis Virus, Infectious Bronchitis Virus and InfectiousBursal Disease Virus.
 9. The recombinant chimeric virus of claim 8,wherein the polypeptide is Marek's Disease Virus glycoprotein A, Marek'sDisease Virus glycoprotein B, or Marek's Disease Virus glycoprotein D.10. The recombinant chimeric virus of claim 8, wherein the polypeptideis a Newcastle Disease virus fusion protein or Newcastle Disease virushemagglutinin-neuraminidase.
 11. The recombinant chimeric virus of claim8, wherein the polypeptide is Infectious Laryngotracheitis Virusglycoprotein B, Infectious Laryngotracheitis Virus glycoprotein I, orInfectious Laryngotracheitis Virus glycoprotein D.
 12. The recombinantchimeric virus of claim 8, wherein the polypeptide is InfectiousBronchitis Virus spike protein or Infectious Bronchitis Virus matrixprotein.
 13. The recombinant chimeric virus of claim 8, wherein thepolypeptide is Infectious Bursal Disease Virus VP2, Infectious BursalDisease Virus VP3 or Infectious Bursal Disease Virus VP4.
 14. Therecombinant chimeric virus of claim 2, wherein the foreign DNA is underthe control of a herpesvirus promoter.
 15. The recombinant chimericvirus of claim 2, wherein the foreign DNA is under the control of aheterologous promoter.
 16. The recombinant chimeric virus of claim 14,wherein the promoter is obtained from an endogenous upstream ofPseudorabies Virus glycoprotein X, Herpesvirus Simplex Virus type--1alpha 4, Human Cytomegalovirus Immediate Early, Marek's Disease Virusglycoprotein A, Marek's Disease Virus glycoprotein B. Marek's DiseaseVirus glycoprotein D, Infectious Laryngotracheitis Virus glycoprotein B,Bovine Herpesvirus 1.1 VP8, or Infectious Laryngotracheitis virusglycoprotein D.
 17. The recombinant chimeric virus of claim 14, whereinthe promoter is obtained from an endogenous upstream sequence ofPseudorabies Virus glycoprotein X, Herpesvirus Simplex Virus type--1alpha 4, Human Cytomegalovirus Immediate Early, Marek's Disease Virusglycoprotein A, Marek's Disease Virus glycoprotein B, Marek's DiseaseVirus glycoprotein D, Infectious Laryngotracheitis Virus glycoprotein B,Bovine Herpresvirus 1.1 VP8, or Infectious Laryngotracheitis virus ofglycoprotein D.
 18. A vaccine which comprises an effective immunizingamount of the recombinant chimeric virus of claim 1 and a suitablecarrier.
 19. A vaccine which comprises an effective immunizing amount ofthe recombinant chimeric virus of claim 2 and a suitable carrier.
 20. Avaccine which comprises an effective immunizing amount of therecombinant chimeric virus of claim 17 and a suitable carrier.
 21. Amethod of immunizing a bird which comprises administering to the bird aneffecting immunizing dose of the vaccine of claim 18.